Activatable interleukin-2 polypeptides

ABSTRACT

The disclosure features fusion proteins that are conditionally active variants of IL-2. In one aspect, the full-length polypeptides of the invention have reduced or minimal cytokine-receptor activating activity even though they contain a functional cytokine polypeptide. Upon activation, e.g., by cleavage of a linker that joins a blocking moiety, e.g., a steric blocking polypeptide, in sequence to the active cytokine, the cytokine can bind its receptor and effect signaling.

RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/US2019/032321, filed May 14, 2019, which claims thebenefit of U.S. Provisional Application 62/671,225, filed on May 14,2018, U.S. Provisional Application No. 62/756,504, filed on Nov. 6,2018, and U.S. Provisional Application No. 62/756,507, filed on Nov. 6,2018. The entire teachings of the above applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 31, 2019, isnamed 105365_0026_SL.txt and is 405,194 bytes in size.

BACKGROUND

The development of mature immunocompetent lymphoid cells fromless-committed precursors, their subsequent antigen-driven immuneresponses, and the suppression of these and unwanted autoreactiveresponses are highly dependent and regulated by cytokines (includinginterleukin-2 [IL-2], IL-4, IL-7, IL-9, IL-15, and IL-21) that utilizereceptors in the common γ-chain (γc) family (Rochman et al., 2009) andfamily members including IL-12, 18 and 23. IL-2 is essential for thymicdevelopment of Treg cells and critically regulates several key aspectsof mature peripheral Treg and antigen-activated conventional T cells.Because of its potent T cell growth factor activity in vitro, IL-2 hasbeen extensively studied in part because this activity offered apotential means to directly boost immunity, e.g., in cancer and AIDS-HIVpatients, or a target to antagonize unwanted responses, e.g.,transplantation rejection and autoimmune diseases. Although in vitrostudies with IL-2 provided a strong rationale for these studies, thefunction of IL-2 in vivo is clearly much more complex as firstillustrated in IL-2-deficient mice, where a rapid lethal autoimmunesyndrome, not lack of immunity, was observed (Sadlack et al., 1993,1995) Similar observations were later made when the gene encoding IL-2Rα(Il2ra) and IL-2Rβ (Il2rb) were individually ablated (Suzuki et al.,1995; Willerford et al., 1995).

The present invention refers to conditionally active and/or targetedcytokines for use in the treatment of cancer and other diseasesdependent on immune up or down regulation. For example, the antitumoralactivity of some cytokines is well known and described and somecytokines have already been used therapeutically in humans. Cytokinessuch as interleukin-2 (IL-2) have shown positive antitumoral activity inpatients with different types of tumors, such as kidney metastaticcarcinoma, hairy cell leukemia, Kaposi sarcoma, melanoma, multiplemyeloma, and the like. Other cytokines like IFNβ, the Tumor NecrosisFactor (TNF) α, TNFβ, IL-1, 4, 6, 12, 15 and the CSFs have shown acertain antitumoral activity on some types of tumors and therefore arethe object of further studies.

SUMMARY

Provided herein are therapeutic proteins, nucleic acids that encode theproteins, and compositions and methods of using the proteins and nucleicacids for the treatment of a disease or disorder, such as proliferativedisease, a tumorous disease, an inflammatory disease, an immunologicaldisorder, an autoimmune disease, an infectious disease, a viral disease,an allergic reaction, a parasitic reaction, graft-versus-host diseaseand the like.

The invention features fusion proteins that are conditionally activevariants of IL-2. In one aspect, the full-length polypeptides of theinvention have reduced or minimal IL-2-receptor activating activity eventhough they contain a functional cytokine polypeptide. Upon activation,e.g., by cleavage of a linker that joins a blocking moiety, e.g., asteric blocking polypeptide, in sequence to the active cytokine, IL-2 orfunctional fragment or mutein thereof, can bind its receptor and effectsignaling. If desired, the full-length polypeptides can include ablocking polypeptide moiety that also provides additional advantageousproperties. For example, the full-length polypeptide can contain ablocking polypeptide moiety that also extends the serum half-life and/ortargets the full-length polypeptide to a desired site of IL-2 activity.Alternatively, the full-length fusion polypeptides can contain a serumhalf-life extension element and/or targeting domain that are distinctfrom the blocking polypeptide moiety. Preferably, the fusion proteincontains at least one element or domain capable of extending in vivocirculating half-life. Preferably, this element is removed enzymaticallyin the desired body location (e.g., protease cleavage in the tumormicroenvironment), restoring pharmacokinetic properties to the payloadmolecule (e.g., IL-2) substantially similar to the naturally occurringpayload molecule. Preferably, the fusion proteins are targeted to adesired cell or tissue. As described herein targeting is accomplishedthrough the action of a blocking polypeptide moiety that also binds to adesired target, or through a targeting domain. The domain thatrecognizes a target antigen on a preferred target (for example atumor-specific antigen), may be attached to the cytokine via a cleavableor non-cleavable linker. If attached by a non-cleavable linker, thetargeting domain may further aid in retaining the cytokine in the tumor,and may be considered a retention domain. The targeting domain does notnecessarily need to be directly linked to the payload molecule, and maybe linked directly to another element of the fusion protein. This isespecially true if the targeting domain is attached via a cleavablelinker.

In one aspect is provided a fusion polypeptide comprising an IL-2polypeptide, or functional fragment or mutein thereof, and a blockingmoiety, e.g., a steric blocking domain. The blocking moiety is fused tothe IL-2 polypeptide, directly or through a linker, and can be separatedfrom the cytokine polypeptide by cleavage (e.g., protease-mediatedcleavage) of the fusion polypeptide at or near the fusion site or linkeror in the blocking moiety. For example, when the cytokine polypeptide isfused to a blocking moiety through a linker that contains a proteasecleavage site, the cytokine polypeptide is released from the blockingmoiety and can bind its receptor, upon protease-mediated cleavage of thelinker. The linker is designed to be cleaved at the site of desiredcytokine activity, for example in the tumor microenvironment, avoidingoff-target cytokine activity and reducing overall toxicity of cytokinetherapy.

In one embodiment, a fusion polypeptide is provided that includes atleast one of each of a human interleukin 2 (IL-2) polypeptide [A], anIL-2 blocking moiety [D], and a protease-cleavable polypeptide linker[L], where the IL-2 polypeptide and the IL-2 blocking moiety areoperably linked by the protease-cleavable polypeptide linker and thefusion polypeptide has attenuated IL-2-receptor activating activity.Typically, the IL-2-receptor activating activity of the fusionpolypeptide is at least about 10 fold less than the IL-2-receptoractivating activity of the polypeptide that contains the IL-2polypeptide that is produced by cleavage of the protease-cleavablelinker.

In another embodiment, a fusion polypeptide is provided that has atleast one of each of a human interleukin 2 (IL-2) polypeptide [A], ahalf-life extension element [B], an IL-2 blocking moiety [D], and aprotease-cleavable polypeptide linker [L], where the IL-2 polypeptideand the IL-2 blocking moiety can be operably linked by theprotease-cleavable polypeptide linker and the fusion polypeptide hasattenuated IL-2-receptor activating activity. Typically, theIL-2-receptor activating activity of the fusion polypeptide is at leastabout 10 fold less than the IL-2-receptor activating activity of thepolypeptide that contains the IL-2 polypeptide that is produced bycleavage of the protease-cleavable linker. The serum half-life of theIL-2 polypeptide that is produced by cleavage of the protease-cleavablepolypeptide linker is typically comparable to the half-life of naturallyoccurring IL-2.

The fusion polypeptide can have the formula:[A]-[L1]-[B]-[L2]-[D],[A]-[L1]-[D]-[L2]-[B],[D]-[L2]-[B]-[L1]-[A],[B]-[L2]-[D]-[L1]-[A],[D]-[L1]-[B]-[L1]-[A],[B]-[L1]-[D]-[L1]-[A],[B]-[L1]-[A]-[L1]-[D], or[D]-[L1]-[A]-[L1]-[B],where A is an interleukin 2 (IL-2) polypeptide; B is a half-lifeextension element; L1 and L2 are each independently a polypeptidelinker, where L1 is a protease-cleavable polypeptide linker and L2 isoptionally a protease-cleavable polypeptide linker; D is an IL-2blocking moiety. In a further embodiment, the fusion polypeptide hasattenuated IL-2-receptor activating activity. In some embodiments, theIL-2-receptor activating activity of the fusion polypeptide is at leastabout 10 fold less than the IL-2-receptor activating activity of thepolypeptide that contains the IL-2 polypeptide that is produced bycleavage of the protease-cleavable polypeptide linker L1.

The fusion polypeptide can further include a tumor-specific antigenbinding peptide. For example, the tumor-specific antigen binding peptideof the fusion polypeptide can be linked to any one of [A], [B], or [D]by a non-cleavable linker. The tumor-specific antigen binding peptidecan be linked to any one of [A], [B], or [D] by a cleavable linker. Thetumor-specific antigen binding peptide of the fusion polypeptide can belinked to the IL-2 polypeptide by a non-cleavable linker and the IL-2polypeptide can be linked to the half-life extension element or the IL-2blocking moiety by a cleavable linker.

The fusion polypeptide can bind IL-2 receptor alpha (IL-2Ra) in a mannersubstantially similar to the naturally occurring IL-2. In someembodiments, the blocking moiety of the fusion polypeptide inhibitsactivation of IL-2 receptor alpha/beta/gamma (IL-2Rαβγ) and IL-2receptor beta/gamma (IL-2Rβγ) by the IL-2 polypeptide in the uncleavedfusion polypeptide.

The IL-2-receptor activating activity of the fusion polypeptide can beassessed, for example, using a CTLL-2 proliferation assay, a phosphoSTAT ELISA, or HEK Blue reporter cell assay and using equal amounts on amole basis of the IL-2 polypeptide and the fusion polypeptide.

The fusion polypeptide may include a plurality of protease-cleavablepolypeptide linkers, where each protease-cleavable polypeptide linkerindependently comprises at least one sequence that is capable of beingcleaved by a protease such as a kallikrein, thrombin, chymase,carboxypeptidase A, cathepsin G, cathepsin L, an elastase, PR-3,granzyme M, a calpain, a matrix metalloproteinase (MMP), a fibroblastactivation protein (FAP), an ADAM metalloproteinase, a plasminogenactivator, a cathepsin, a caspase, a tryptase, or a tumor cell surfaceprotease. Each protease-cleavable polypeptide of the fusion polypeptidecan independently comprise two or more cleavage sites for the sameprotease, or two or more cleavage sites that can be cleaved by differentproteases, or at least one of the protease-cleavable polypeptides cancomprises a cleavage site for two or more different proteases.

In some embodiments, the IL-2 blocking moiety of the fusion polypeptidesof the invention inhibits activation of the IL-2 receptor by the fusionpolypeptide. In some embodiments, the IL-2 blocking moiety can comprise,for example, a ligand-binding domain or fragment of a cognate receptorfor the IL-2, a single domain antibody, Fab or scFv that binds the IL-2polypeptide, or an antibody or antibody fragment that binds a receptorof the IL-2.

The half-life extension element of the fusion polypeptide can be, forexample, human serum albumin, an antigen-binding polypeptide that bindshuman serum albumin, or an immunoglobulin Fc.

In some embodiments, the blocking moiety can also function as a serumhalf-life extension element. In some other embodiments, the fusionpolypeptide further comprises a separate serum half-life extensionelement. In some embodiments, the fusion polypeptide further comprises atargeting domain. In various embodiments, the serum half-life extensionelement is a water-soluble polypeptide such as optionally branched ormulti-armed polyethylene glycol (PEG), full length human serum albumin(HSA) or a fragment that preserves binding to FcRn, an Fc fragment, or ananobody that binds to FcRn directly or to human serum albumin.

In addition to serum half-life extension elements, the pharmaceuticalcompositions described herein preferably comprise at least one, or moretargeting domains that bind to one or more target antigens or one ormore regions on a single target antigen. It is contemplated herein thata polypeptide construct of the invention is cleaved, for example, in adisease-specific microenvironment or in the blood of a subject at theprotease cleavage site and that the targeting domain(s) will bind to atarget antigen on a target cell. At least one target antigen is involvedin and/or associated with a disease, disorder or condition. Exemplarytarget antigens include those associated with a proliferative disease, atumorous disease, an inflammatory disease, an immunological disorder, anautoimmune disease, an infectious disease, a viral disease, an allergicreaction, a parasitic reaction, a graft-versus-host disease or ahost-versus-graft disease.

In some embodiments, a target antigen is a cell surface molecule such asa protein, lipid or polysaccharide. In some embodiments, a targetantigen is a on a tumor cell, virally infected cell, bacteriallyinfected cell, damaged red blood cell, arterial plaque cell, or fibrotictissue cell.

Target antigens, in some cases, are expressed on the surface of adiseased cell or tissue, for example a tumor or a cancer cell. Targetantigens for tumors include but are not limited to Fibroblast activationprotein alpha (FAPa), Trophoblast glycoprotein (5T4), Tumor-associatedcalcium signal transducer 2 (Trop2), Fibronectin EDB (EDB-FN),fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, c-Met,FOLR1, and CEA. Pharmaceutical compositions disclosed herein, alsoinclude proteins comprising two antigen binding domains that bind to twodifferent target antigens known to be expressed on a diseased cell ortissue. Exemplary pairs of antigen binding domains include but are notlimited to EGFR/CEA, EpCAM/CEA, and HER-2/HER-3.

In some embodiments, the targeting polypeptides independently comprise ascFv, a VH domain, a VL domain, a non-Ig domain, or a ligand thatspecifically binds to the target antigen. In some embodiments, thetargeting polypeptides specifically bind to a cell surface molecule. Insome embodiments, the targeting polypeptides specifically bind to atumor antigen. In some embodiments, the targeting polypeptidesspecifically and independently bind to a tumor antigen selected from atleast one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In someembodiments, the targeting polypeptides specifically and independentlybind to two different antigens, wherein at least one of the antigens isa tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, andFOLR1. In some embodiments, the targeting polypeptide serves as aretention domain and is attached to the cytokine via a non-cleavablelinker.

As described herein, the cytokine blocking moiety can bind to IL-2 andthereby block activation of the IL-2 cognate receptor.

This disclosure also related to nucleic acids, e.g., DNA, RNA, mRNA,that encode the conditionally active proteins described herein, as wellas vectors and host cells that contain such nucleic acids.

This disclosure also relates to pharmaceutical compositions that containa conditionally active protein, nucleic acid that encodes theconditionally active protein, and vectors and host cells that containsuch nucleic acids. Typically, the pharmaceutical composition containsone or more physiologically acceptable carriers and/or excipients. Thedisclosure also relates to methods of making a pharmaceuticalcomposition that include culturing host cell that contain nucleic acidsencoding the fusion polypeptides of the invention under suitableconditions for expression and collection of the fusion polypeptides.

The disclosure also relates to therapeutic methods that includeadministering to a subject in need thereof an effective amount of aconditionally active protein, nucleic acid that encodes theconditionally active protein, vector or host cells that contain such anucleic acid, and pharmaceutical compositions of any of the foregoing.Typically, the subject has, or is at risk of developing, a proliferativedisease, a tumorous disease, an inflammatory disease, an immunologicaldisorder, an autoimmune disease, an infectious disease, a viral disease,an allergic reaction, a parasitic reaction, a graft-versus-host diseaseor a host-versus-graft disease.

The disclosure further relates methods for treating a tumor or cancerthat include administering to a subject in need thereof an effectiveamount of a fusion polypeptide of the invention. In some embodiments,the method for treating a tumor or cancer can include administeringeffective amount of the fusion polypeptide intravenously. In someembodiments, the method can further include administration of anadditional chemotherapeutic agent.

The disclosure also relates to the use of a conditionally activeprotein, nucleic acid that encodes the conditionally active protein,vector or host cells that contain such a nucleic acid, andpharmaceutical compositions of any of the foregoing, for treating asubject in need thereof. Typically the subject has, or is at risk ofdeveloping, a proliferative disease, a tumorous disease, an inflammatorydisease, an immunological disorder, an autoimmune disease, an infectiousdisease, a viral disease, an allergic reaction, a parasitic reaction, agraft-versus-host disease or a host-versus-graft disease.

The disclosure also relates to the use of a conditionally activeprotein, nucleic acid that encodes the conditionally active protein,vector or host cells that contain such a nucleic acid for themanufacture of a medicament for treating a disease, such as aproliferative disease, a tumorous disease, an inflammatory disease, animmunological disorder, an autoimmune disease, an infectious disease, aviral disease, an allergic reaction, a parasitic reaction, agraft-versus-host disease or a host-versus-graft disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a schematic illustrating a protease-activated cytokine orchemokine that includes a blocking moiety. The blocking moiety mayoptionally function as a serum half-life extending domain. To the leftof the arrow the drawing shows that a cytokine is connected to ablocking moiety via a protease-cleavable linker, thus blocking itsability to bind to its receptor. To the right of the arrow the drawingshows that in an inflammatory or tumor environment a protease cleaves ata protease-cleavage site on the linker, releasing the blocking moietyand allowing the cytokine to bind to its receptor.

FIG. 1b is a schematic illustrating a protease-activated cytokine orchemokine wherein HSA (blocking moiety) is directly bound to thecytokine or chemokine of interest, with a protease cleavage site betweenthe HSA and a cytokine or chemokine of interest. To the left of thearrow the drawing shows that a cytokine is connected to a blockingmoiety via a protease-cleavable linker, thus blocking its ability tobind to its receptor. To the right of the arrow the drawing shows thatin an inflammatory or tumor environment, the protease cleaves at aprotease-cleavage site on linker, releasing the blocking moiety andallowing the cytokine to bind to its receptor.

FIG. 1c is a schematic illustrating a protease-activated cytokine orchemokine wherein more than one HSA (blocking moiety) is bound directlyto the molecule of interest. If desired, one or more of the HSA can bebonded to the cytokine or chemokine through a linker, such as a linkerthat contains a protease cleavage site. To the left of the arrow thedrawing shows that a cytokine is connected to a blocking moiety via aprotease-cleavable linker, thus blocking its ability to bind to itsreceptor. To the right of the arrow the drawing shows that in aninflammatory or tumor environment, protease cleaves at protease-cleavagesite on linker, releasing the blocking moiety and allowing cytokine tobind receptor. The cytokine now has similar pK properties as compared tothe native cytokine (e.g., has a short half-life).

FIG. 1d is a schematic illustrating a protease-activated cytokine orchemokine comprising more than one cytokine, of the same type ordifferent type, each of which is bonded to a binding domain through aprotease-cleavable linker. To the left of the arrow the drawing showsthat a cytokine is connected to a blocking moiety via aprotease-cleavable linker, thus blocking its ability to bind to itsreceptor. To the right of the arrow the drawing shows that in aninflammatory or tumor environment a protease cleaves at a proteasecleavage site on linker, releasing the blocking moiety and allowing thecytokine to bind to its receptor.

FIG. 2 is a schematic illustrating a protease-activated cytokine orchemokine comprising a cytokine or chemokine polypeptide, a blockingmoiety, and a serum half-life extending domain connected by at least oneprotease-cleavable linker. To the left of the arrow the drawing showsthat a cytokine is connected to a blocking moiety via protease-cleavablelinkers, thus blocking its ability to bind to its receptor. It is alsobound to a separate half-life extension element, which extends half-lifein serum. To the right of the arrow the drawing shows that in aninflammatory or tumor environment a protease cleaves at aprotease-cleavage site on linker, thus releasing the serum half-lifeextension element and the blocking moiety and allowing the cytokine tobind to its receptor. The cytokine now has similar pK properties ascompared to the native cytokine (e.g., a short half-life).

FIG. 3 is a schematic illustrating a protease-activated cytokine orchemokine comprising a cytokine or chemokine polypeptide, a blockingmoiety, and a targeting domain connected by at least oneprotease-cleavable linker. To the left of the arrow the drawing showsthat a cytokine is connected to a blocking moiety and a targeting domainvia a protease-cleavable linker, thus blocking its ability to bind toits receptor. To the right of the arrow the drawing shows that in aninflammatory or tumor microenvironment a protease cleaves at theprotease cleavage site in the linker, releasing the targeting domain andthe blocking moiety and allowing the cytokine to bind to its receptor.

FIG. 4a is a schematic illustrating a protease-activated cytokine orchemokine comprising a cytokine or chemokine polypeptide, a blockingmoiety, a targeting domain, and a serum half-life extending domainconnected by at least one protease-cleavable linker, wherein thecytokine polypeptide and the targeting domain are connected by aprotease-cleavable linker. To the left of the arrow, the drawing showsthat a cytokine is connected to targeting domain, blocking moiety, andhalf-life extension element via protease-cleavable linker(s), thusblocking its ability to bind to its receptor. To the right of the arrowthe drawing shows that in an inflammatory or tumor environment, theprotease cleaves at a protease-cleavage site on linker(s), releasing thehalf-life extension element, the targeting domain, and the blockingmoiety, and allowing the cytokine to bind to its receptor. The cytokinenow has similar pK properties as compared to the native cytokine (e.g.,short half-life).

FIG. 4b is a schematic illustrating a protease-activated cytokine orchemokine comprising a cytokine or chemokine polypeptide, a blockingmoiety, a targeting domain, and a serum half-life extending domainconnected by at least one protease-cleavable linker. To the left of thearrow, the drawing shows that a cytokine is connected to targetingdomain, a blocking moiety, and a half-life extension element viaprotease-cleavable linker(s), thus blocking its ability to bind to itsreceptor. To the right of the arrow the drawing shows that in aninflammatory or tumor environment, the protease cleaves at aprotease-cleavage site on linker(s), releasing the half-life extensionelement and the blocking moiety and allowing the cytokine to bind to thereceptor. The targeting moiety remains bound, keeping the cytokine inthe tumor microenvironment. The cytokine now has similar pK propertiesas compared to the native cytokine (e.g., a short half-life).

FIG. 5 is a schematic illustrating the structure of a variable domain ofan immunoglobulin molecule. The variable domains of both light and heavyimmunoglobulin chains contain three hypervariable loops, orcomplementarity-determining regions (CDRs). The three CDRs of a V domain(CDR1, CDR2, CDR3) cluster at one end of the beta barrel. The CDRs arethe loops that connect beta strands B-C, C′-C″, and F-G of theimmunoglobulin fold, whereas the bottom loops that connect beta strandsAB, CC′, C″-D and E-F of the immunoglobulin fold, and the top loop thatconnects the D-E strands of the immunoglobulin fold are the non-CDRloops.

FIG. 6 is a schematic illustrating a protease-activated cytokine orchemokine comprising a cytokine or chemokine polypeptide, a blockingmoiety that is a serum albumin binding domain (e.g., a dAb), and aprotease-cleavable linker. In the illustrated example, the non-CDR loopsin a serum albumin binding domain (e.g., a sdAb) can form a binding sitefor the cytokine IL-2. In this example, the binding site for serumalbumin can be formed by the CDRs of the serum albumin binding domain.

FIGS. 7a-7h are a series of graphs showing activity of exemplary IL-2fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell lineCTLL-2. Each graph shows results of the IL-2 proliferation assay asquantified by CellTiter-Glo® (Promega) luminescence-based cell viabilityassay. Each proliferation assay was performed with HSA (FIG. 7b , FIG.7d , FIG. 7f , and FIG. 7h ) or without (FIG. 7a , FIG. 7c , FIG. 7e ,and FIG. 7g ). Each fusion protein comprises an anti-HSA binder, andboth uncleaved and MMP9 protease cleaved versions of the fusion proteinwere used in each assay.

FIGS. 8a-8f are a series of graphs showing activity of exemplary IL-2fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell lineCTLL-2. Each graph shows results of the IL-2 proliferation assay asquantified by CellTiter-Glo (Promega) luminescence-based cell viabilityassay. Both uncleaved and MMP9 protease cleaved versions of the fusionprotein were used in each assay.

FIGS. 9a-9z are a series of graphs showing activity of exemplary IL-2fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell lineCTLL-2. Each graph shows results of the IL-2 proliferation assay asquantified by CellTiter-Glo (Promega) luminescence-based cell viabilityassay. Both uncleaved and MMP9 protease cleaved versions of the fusionprotein were used in each assay.

FIG. 10 shows results of protein cleavage assay, as described in Example2. Fusion protein ACP16 was run on an SDS-PAGE gel in both cleaved anduncleaved form. As can be seen in the gel, cleavage was complete.

FIGS. 11a-11g is a series of graphs depicting results from a HEK-BlueIL-2 reporter assay performed on IL-2 fusion proteins and recombinanthuman IL-2 (Rec hIL-2) (FIGS. 11a, 11c, 11e , and 11f) or cleavage ofthe fusion proteins shown in SDS-PAGE gels (FIGS. 11b and 11d ).Analysis was performed based on quantification of Secreted AlkalinePhosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen).FIG. 11g is a schematic showing the structure of the fusion proteinACP16.

FIG. 12a and FIG. 12b are two graphs showing analysis of ACP16 (an IL-2fusion protein) (FIG. 12a ) and ACP124 (a non-cleavable IL-2 fusionprotein) (FIG. 12b ) in a HEK Blue IL-2 reporter assay in the presenceof HSA. Circles depict the activity of the uncut polypeptide, squaresdepict activity of the cut polypeptide. FIG. 12c is a graph showingresults of a CTLL-2 proliferation assay. CTLL2 cells (ATCC) were platedin suspension at a concentration of 500,000 cells/well in culture mediawith or without 40 mg/ml human serum albumin (HSA) and stimulated with adilution series of activatable hIL-2 for 72 hours at 37° C. and 5% CO₂.Activity of uncleaved and cleaved activatable ACP16 was tested. Cleavedactivatable hIL-2 was generated by incubation with active MMP9. Cellactivity was assessed using a CellTiter-Glo (Promega) luminescence-basedcell viability assay. Circles depict intact fusion protein, and squaresdepict protease-cleaved fusion protein.

FIGS. 13a-13c are graphs showing results of analyzing ACP16 and ACP124in a tumor xenograft model. FIG. 13a shows tumor volume over time inmice treated with 4.4 μg ACP16 (squares), 17 μg ACP16 (triangles), 70 μgACP16 (downward triangles), 232 μg ACP16 (dark circles), and as acomparator, 12 μg wild type IL-2 (dashed line, triangles) and 36 μg wildtype IL-2 (dashed line, diamonds). Vehicle alone is indicated by largeopen circles. The data show tumor volume decreasing over time in adose-dependent manner in mice treated with ACP16 at higherconcentrations. FIG. 13b shows tumor volume over time in mice treatedwith 17 μg ACP124 (squares), 70 μg ACP124 (triangles), 230 μg ACP124(downward triangles), and 700 μg ACP124. Vehicle alone is indicated bylarge open circles. FIG. 13c shows tumor volume over time in micetreated with 17 μg ACP16 (triangles), 70 μg ACP16 (circles), 232 μgACP16 (dark circles), and as a comparator 17 μg ACP124 (dashed line,triangles) 70 μg ACP124 (dashed line, diamonds), 230 μg ACP124 (dashedline, diamonds). Vehicle alone is indicated by dark downward triangles.The data show tumor volume decreasing over time in a dose-dependentmanner in mice treated with ACP16, but not ACP124.

FIGS. 14a-14i are a series of “spaghetti” plots showing activity offusion proteins in an MC38 mouse xenograft model corresponding to thedata shown in FIGS. 13a-13c . Each line in the plots represents a singlemouse. Shown are vehicle alone (FIG. 14a ), 4.4, 17, 70, and 232 μgACP16 (FIG. 14b , FIG. 14c , FIG. 14d , and FIG. 14e ), and 17, 70, 230,and 700 μg ACP124 (FIG. 14f , FIG. 14g , FIG. 14h , and FIG. 14i ).

FIG. 15 is a graph showing tumor volume over time in a mouse xenograftmodel showing tumor growth in control mice (open circles) andAP16-treated mice (squares).

FIGS. 16a-16d are a series of survival plots showing survival of miceover time after treatment with cleavable fusion proteins. FIG. 16a showsdata for mice treated with vehicle alone (gray line), 17 μg ACP16 (darkline), and 17 μg ACP124 (dashed line). FIG. 16b shows data for micetreated with vehicle alone (gray line), 70 μg ACP16 (dark line), and 70μg ACP124 (dashed line). FIG. 16c shows data for mice treated withvehicle alone (gray line), 232 μg ACP16 (dark line), and 230 μg ACP124(dashed line). FIG. 16d shows data for mice treated with vehicle alone(gray line), 232 μg ACP16 (dark line), and 700 μg ACP124 (dashed line).

FIG. 17a-17m are a series of “spaghetti” plots showing activity offusion proteins in an MC38 mouse xenograft model. All mouse groups weregiven four doses total except for the highest three doses of APC132,wherein fatal toxicity was detected after 1 week/2 doses. Shown arevehicle alone (FIG. 17a ), 17, 55, 70, and 230 μg ACP16 (FIG. 17b , FIG.17c , FIG. 17d , and FIG. 17e ), 9, 28, 36, and 119 μg ACP132 (FIG. 17f, FIG. 17g , FIG. 17h , and FIG. 17i ), and 13, 42, 54, and 177 μg ACP21(FIG. 17j , FIG. 17k , FIG. 17l , and FIG. 17m ). Each line in the plotsrepresents an individual animal.

FIG. 18 illustrates the properties of ProTriTac polypeptides, whichserve as exemplary protease cleavable fusion proteins.

FIG. 19 illustrates differential activities of ProTriTAC proteinsmeasured by ELISA, flow cytometry, and T cell-dependent cellularcytotoxicity assay.

FIG. 20 illustrates ProTriTAC exhibits potent, protease-dependent,anti-tumor activity in a rodent tumor xenograft model.

FIG. 21 illustrates SDS-PAGE analysis of purified ProTriTAC proteins.

FIG. 22 illustrates analytical SEC of a ProTriTAC protein afterdifferent stress conditions.

FIG. 23 demonstrates functional masking and stability of ProTriTAC incynomolgus monkey pharmacokinetic study.

DETAILED DESCRIPTION

Disclosed herein are methods and compositions to engineer and useconstructs comprising inducible cytokines. Cytokines are potent immuneagonists, which lead to them being considered promising therapeuticagents for oncology. However, cytokines proved to have a very narrowtherapeutic window. Cytokines have short serum half-lives and are alsoconsidered to be highly potent. Consequently, therapeutic administrationof cytokines produced undesirable systemic effects and toxicities. Thesewere exacerbated by the need to administer large quantities of cytokinein order to achieve the desired levels of cytokine at the intended siteof cytokine action (e.g., a tumor). Unfortunately, due to the biology ofcytokines and inability to effectively target and control theiractivity, cytokines did not achieve the hoped for clinical advantages inthe treatment of tumors.

Disclosed herein are fusion proteins that overcome the toxicity andshort half-life problems that have severely limited the clinical use ofcytokines in oncology. The fusion proteins contain cytokine polypeptidesthat have receptor agonist activity. But in the context of the fusionprotein, the cytokine receptor agonist activity is attenuated and thecirculating half-life is extended. The fusion proteins include proteasecleave sites, which are cleaved by proteases that are associated with adesired site of cytokine activity (e.g., a tumor), and are typicallyenriched or selectively present at the site of desired activity. Thus,the fusion proteins are preferentially (or selectively) and efficientlycleaved at the desired site of activity to limit cytokine activitysubstantially to the desired site of activity, such as the tumormicroenvironment. Protease cleavage at the desired site of activity,such as in a tumor microenvironment, releases a form of the cytokinefrom the fusion protein that is much more active as a cytokine receptoragonist than the fusion protein (typically at least about 100× moreactive than the fusion protein). The form of the cytokine that isreleased upon cleavage of the fusion protein typically has a shorthalf-life, which is often substantially similar to the half-life of thenaturally occurring cytokine, further restricting cytokine activity tothe tumor microenvironment. Even though the half-life of the fusionprotein is extended, toxicity is dramatically reduced or eliminatedbecause the circulating fusion protein is attenuated and active cytokineis targeted to the tumor microenvironment. The fusion proteins describedherein, for the first time, enable the administration of an effectivetherapeutic dose of a cytokine to treat tumors with the activity of thecytokine substantially limited to the tumor microenvironment, anddramatically reduces or eliminates unwanted systemic effects andtoxicity of the cytokine.

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a difference over what is generally understood in the art. Thetechniques and procedures described or referenced herein are generallywell understood and commonly employed using conventional methodologiesby those skilled in the art, such as, for example, the widely utilizedmolecular cloning methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 4th ed. (2012) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. As appropriate, proceduresinvolving the use of commercially available kits and reagents aregenerally carried out in accordance with manufacturer-defined protocolsand conditions unless otherwise noted.

“Cytokine” is a well-known term of art that refers to any of a class ofimmunoregulatory proteins (such as interleukin or interferon) that aresecreted by cells especially of the immune system and that aremodulators of the immune system. Cytokine polypeptides that can be usedin the fusion proteins disclosed herein include, but are not limited totransforming growth factors, such as TGF-α and TGF-β (e.g., TGFbeta1,TGFbeta2, TGFbeta3); interferons, such as interferon-α, interferon-β,interferon-γ, interferon-kappa and interferon-omega; interleukins, suchas IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-25;tumor necrosis factors, such as tumor necrosis factor alpha andlymphotoxin; chemokines (e.g., C-X-C motif chemokine 10 (CXCL10), CCL19,CCL20, CCL21), and granulocyte macrophage-colony stimulating factor(GM-CS), as well as fragments of such polypeptides that active thecognate receptors for the cytokine (i.e., functional fragments of theforegoing). “Chemokine” is a term of art that refers to any of a familyof small cytokines with the ability to induce directed chemotaxis innearby responsive cells.

Cytokines are well-known to have short serum half-lives that frequentlyare only a few minutes or hours. Even forms of cytokines that havealtered amino acid sequences intended to extend the serum half-life yetretain receptor agonist activity typically also have short serumhalf-lives. As used herein, a “short-half-life cytokine” refers to acytokine that has a substantially brief half-life circulating in theserum of a subject, such as a serum half-life that is less than 10, lessthan 15, less than 30, less than 60, less than 90, less than 120, lessthan 240, or less than 480 minutes. As used herein, a short half-lifecytokine includes cytokines which have not been modified in theirsequence to achieve a longer than usual half-life in the body of asubject and polypeptides that have altered amino acid sequences intendedto extend the serum half-life yet retain receptor agonist activity.Typically a short half-life cytokine polypeptide, such as an IL-2polypeptide has a serum half-life that is comparable to naturallyoccurring IL-2, e.g., within 5 fold, 4 fold, 3 fold or 2 fold ofnaturally occurring IL-2. This latter case is not meant to include theaddition of heterologous protein domains, such as a bona fide half-lifeextension element, such as serum albumin.

“Sortases” are transpeptidases that modify proteins by recognizing andcleaving a carboxyl-terminal sorting signal embedded in or terminallyattached to a target protein or peptide. Sortase A catalyzes thecleavage of the LPXTG motif (SEQ ID NO: 125) (where X is any standardamino acid) between the Thr and Gly residue on the target protein, withtransient attachment of the Thr residue to the active site Cys residueon the enzyme, forming an enzyme-thioacyl intermediate. To completetranspeptidation and create the peptide-monomer conjugate, a biomoleculewith an N-terminal nucleophilic group, typically an oligoglycine motif,attacks the intermediate, displacing Sortase A and joining the twomolecules.

As used herein, the term “steric blocker” refers to a polypeptide orpolypeptide moiety that can be covalently bonded to a cytokinepolypeptide directly or indirectly through other moieties such aslinkers, for example in the form of a chimeric polypeptide (fusionprotein), but otherwise does not covalently bond to the cytokinepolypeptide. A steric blocker can non-covalently bond to the cytokinepolypeptide, for example though electrostatic, hydrophobic, ionic orhydrogen bonding. A steric blocker typically inhibits or blocks theactivity of the cytokine moiety due to its proximity to the cytokinemoiety and comparative size. A steric blocker may also block by virtueof recruitment of a large protein binding partner. An example of this isan antibody, which binds to serum albumin; while the antibody itself mayor may not be large enough to block activation or binding on its own,recruitment of albumin allows for sufficient steric blocking.

As used herein, the term “operably linked” in the context of a fusionpolypeptide refers to orientation of the components of a fusionpolypeptide that permits the components to function in their intendedmanner. For example, an IL-2 polypeptide and an IL-2 blocking moiety areoperably linked by a protease-cleavable polypeptide linker in a fusionpolypeptide when the IL-2 blocking moiety is capable of inhibiting theIL-2 receptor-activating activity of the IL-2 polypeptide in the fusionpolypeptide, for example by binding to the IL-2 polypeptide, but uponcleavage of the protease-cleavable polypeptide linker the inhibition ofthe IL-2 receptor-activating activity of the IL-2 polypeptide by theIL-2 blocking moiety is decreased or eliminated, for example because theIL-2 blocking moiety can diffuse away from the IL-2 polypeptide.

As used and described herein, a “half-life extension element” is a partof the chimeric polypeptide that increases the serum half-life andimprove pK, for example, by altering its size (e.g., to be above thekidney filtration cutoff), shape, hydrodynamic radius, charge, orparameters of absorption, biodistribution, metabolism, and elimination.

As used herein, the terms “activatable,” “activate,” “induce,” and“inducible” refer to the ability of a protein, i.e. a cytokine, that ispart of a fusion protein, to bind its receptor and effectuate activityupon cleavage of additional elements from the fusion protein.

As used herein, “plasmids” or “viral vectors” are agents that transportthe disclosed nucleic acids into the cell without degradation andinclude a promoter yielding expression of the nucleic acid moleculeand/or polypeptide in the cells into which it is delivered.

As used herein, the terms “peptide”, “polypeptide”, or “protein” areused broadly to mean two or more amino acids linked by a peptide bond.Protein, peptide, and polypeptide are also used herein interchangeablyto refer to amino acid sequences. It should be recognized that the termpolypeptide is not used herein to suggest a particular size or number ofamino acids comprising the molecule and that a peptide of the inventioncan contain up to several amino acid residues or more.

As used throughout, “subject” can be a vertebrate, more specifically amammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse,rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and anyother animal. The term does not denote a particular age or sex. Thus,adult and newborn subjects, whether male or female, are intended to becovered.

As used herein, “patient” or “subject” may be used interchangeably andcan refer to a subject with a disease or disorder (e.g., cancer). Theterm patient or subject includes human and veterinary subjects.

As used herein the terms “treatment”, “treat”, or “treating” refers to amethod of reducing the effects of a disease or condition or symptom ofthe disease or condition. Thus, in the disclosed method, treatment canrefer to at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, or substantially completereduction in the severity of an established disease or condition orsymptom of the disease or condition. For example, a method for treatinga disease is considered to be a treatment if there is a 10% reduction inone or more symptoms of the disease in a subject as compared to acontrol. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or any percent reduction in between 10% and 100% ascompared to native or control levels. It is understood that treatmentdoes not necessarily refer to a cure or complete ablation of thedisease, condition, or symptoms of the disease or condition.

As used herein, the terms “prevent”, “preventing”, and “prevention” of adisease or disorder refers to an action, for example, administration ofthe chimeric polypeptide or nucleic acid sequence encoding the chimericpolypeptide, that occurs before or at about the same time a subjectbegins to show one or more symptoms of the disease or disorder, whichinhibits or delays onset or exacerbation of one or more symptoms of thedisease or disorder.

As used herein, references to “decreasing”, “reducing”, or “inhibiting”include a change of at least about 10%, of at least about 20%, of atleast about 30%, of at least about 40%, of at least about 50%, of atleast about 60%, of at least about 70%, of at least about 80%, of atleast about 90% or greater as compared to a suitable control level. Suchterms can include but do not necessarily include complete elimination ofa function or property, such as agonist activity.

An “attenuated cytokine receptor agonist” is a cytokine receptor agonistthat has decreased receptor agonist activity as compared to the cytokinereceptor's naturally occurring agonist. An attenuated cytokine agonistmay have at least about 10×, at least about 50×, at least about 100×, atleast about 250×, at least about 500×, at least about 1000× or lessagonist activity as compared to the receptor's naturally occurringagonist. When a fusion protein that contains a cytokine polypeptide asdescribed herein is described as “attenuated” or having “attenuatedactivity”, it is meant that the fusion protein is an attenuated cytokinereceptor agonist.

An “intact fusion protein” is a fusion protein in which no domain hasbeen removed, for example by protease cleavage. A domain may beremovable by protease cleavage or other enzymatic activity, but when thefusion protein is “intact”, this has not occurred.

As used herein “moiety” refers to a portion of a molecule that has adistinct function within that molecule, and that function may beperformed by that moiety in the context of another molecule. A moietymay be a chemical entity with a particular function, or a portion of abiological molecule with a particular function. For example, a “blockingmoiety” within a fusion protein is a portion of the fusion protein whichis capable of blocking the activity of some or all of the fusionpolypeptide. This may be a protein domain, such as serum albumin.Blocking may be accomplished by a steric blocker or a specific blocker.A steric blocker blocks by virtue of size and position and not basedupon specific binding; an examples is serum albumin. A specific blockerblocks by virtue of specific interactions with the moiety to be blocked.A specific blocker must be tailored to the particular cytokine or activedomain; a steric blocker can be used regardless of the payload, as longas it is large enough.

In general, the therapeutic use of cytokines is strongly limited bytheir systemic toxicity. TNF, for example, was originally discovered forits capacity of inducing the hemorrhagic necrosis of some tumors, andfor its in vitro cytotoxic effect on different tumoral lines, but itsubsequently proved to have strong pro-inflammatory activity, which can,in case of overproduction conditions, dangerously affect the human body.As the systemic toxicity is a fundamental problem with the use ofpharmacologically active amounts of cytokines in humans, novelderivatives and therapeutic strategies are now under evaluation, aimedat reducing the toxic effects of this class of biological effectorswhile keeping their therapeutic efficacy.

IL-2 exerts both stimulatory and regulatory functions in the immunesystem and is, along with other members of the common γ chain (γc)cytokine family, central to immune homeostasis. IL-2 mediates its actionby binding to IL-2 receptors (IL-2R), consisting of either trimericreceptors made of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132)chains or dimeric βγ IL-2Rs (1, 3). Both IL-2R variants are able totransmit signal upon IL-2 binding. However, trimeric αβγ IL-2Rs have aroughly 10-100 times higher affinity for IL-2 than dimeric βγ IL-2Rs(3), implicating that CD25 confers high-affinity binding of IL-2 to itsreceptor but is not crucial for signal transduction. Trimeric IL-2Rs arefound on activated T cells and CD4+ forkhead box P3 (FoxP3)+T regulatorycells (Treg), which are sensitive to IL-2 in vitro and in vivo.Conversely, antigen-experienced (memory) CD8+, CD44 highmemory-phenotype (MP) CD8+, and natural killer (NK) cells are endowedwith high levels of dimeric βγ IL-2Rs and these cells also respondvigorously to IL-2 in vitro and in vivo.

Expression of the high-affinity IL-2R is critical for endowing T cellsto respond to low concentrations of IL-2 that is transiently availablein vivo. IL-2Rα expression is absent on naive and memory T cells but isinduced after antigen activation. IL-2Rβ is constitutively expressed byNK, NKT, and memory CD8+ T cells but is also induced on naive T cellsafter antigen activation. γc is much less stringently regulated and isconstitutively expressed by all lymphoid cells. Once the high-affinityIL-2R is induced by antigen, IL-2R signaling upregulates the expressionof IL-2Rα in part through Stat5-dependent regulation of Il2ratranscription (Kim et al., 2001). This process represents a mechanism tomaintain expression of the high-affinity IL-2R and sustain IL-2signaling while there remains a source of IL-2.

IL-2 is captured by IL-2Rα through a large hydrophobic binding surfacesurrounded by a polar periphery that results in a relatively weakinteraction (Kd 10-8 M) with rapid on-off binding kinetics. However, theIL-2Rα-IL-2 binary complex leads to a very small conformational changein IL-2 that promotes association with IL-2Rβ through a distinct polarinteraction between IL-2 and IL-2Rβ. The pseudo-high affinity of theIL-2/α/β trimeric complex (i.e. Kd ˜300 pM) clearly indicates that thetrimeric complex is more stable than either IL-2 bound to the α chainalone (Kd=10 nM) or to the β chain alone (Kd=450 nM) as shown byCiardelli's data. In any event, the IL-2/α/β trimer then recruits the γchain into the quaternary complex capable of signaling, which isfacilitated by the large composite binding site on the IL-2-bound βchain for the γ chain.

In other words, the ternary IL-2Rα-IL-2Rβ-IL-2 complex then recruits γcthrough a weak interaction with IL-2 and a stronger interaction withIL-2Rβ to produce a stable quaternary high-affinity IL-2R (Kd 10-11 Mwhich is 10 pM). The formation of the high-affinity quaternaryIL-2-IL-2R complex leads to signal transduction through the tyrosinekinases Jak1 and Jak3, which are associated with IL-2Rβ and γc,respectively (Nelson and Willerford, 1998). The quaternary IL-2-IL-2Rcomplex is rapidly internalized, where IL-2, IL-2Rβ, and γc are rapidlydegraded, but IL-2Rα is recycled to the cell surface (Hémar et al.,1995; Yu and Malek, 2001). Thus, those functional activities thatrequire sustained IL-2R signaling require a continued source of IL-2 toengage IL-2Rα and form additional IL-2-IL-2R signaling complexes.

Regulatory T cells actively suppress activation of the immune system andprevent pathological self-reactivity and consequent autoimmune disease.Developing drugs and methods to selectively activate regulatory T cellsfor the treatment of autoimmune disease is the subject of intenseresearch and, until the development of the present invention, which canselectively deliver active interleukins at the site of inflammation, hasbeen largely unsuccessful. Regulatory T cells (Treg) are a class ofCD4+CD25+ T cells that suppress the activity of other immune cells. Tregare central to immune system homeostasis, and play a major role inmaintaining tolerance to self-antigens and in modulating the immuneresponse to foreign antigens. Multiple autoimmune and inflammatorydiseases, including Type 1 Diabetes (T1D), Systemic Lupus Erythematosus(SLE), and Graft-versus-Host Disease (GVHD) have been shown to have adeficiency of Treg cell numbers or Treg function.

Consequently, there is great interest in the development of therapiesthat boost the numbers and/or function of Treg cells. One treatmentapproach for autoimmune diseases being investigated is thetransplantation of autologous, ex vivo-expanded Treg cells (Tang, Q., etal, 2013, Cold Spring Harb. Perspect. Med., 3:1-15). While this approachhas shown promise in treating animal models of disease and in severalearly stage human clinical trials, it requires personalized treatmentwith the patient's own T cells, is invasive, and is technically complex.Another approach is treatment with low dose Interleukin-2 (IL-2). Tregcells characteristically express high constitutive levels of the highaffinity IL-2 receptor, IL-2Rαβγ, which is composed of the subunitsIL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132), and Treg cell growthhas been shown to be dependent on IL-2 (Malek, T. R., et al., 2010,Immunity, 33:153-65).

Conversely, immune activation has also been achieved using IL-2, andrecombinant IL-2 (Proleukin®) has been approved to treat certaincancers. High-dose IL-2 is used for the treatment of patients withmetastatic melanoma and metastatic renal cell carcinoma with a long-termimpact on overall survival.

Clinical trials of low-dose IL-2 treatment of chronic GVHD (Koreth, J.,et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmunevasculitis patients (Saadoun, D., et al., 2011, N Engl J Med.,365:2067-77) have demonstrated increased Treg levels and signs ofclinical efficacy. New clinical trials investigating the efficacy ofIL-2 in multiple other autoimmune and inflammatory diseases have beeninitiated. The rationale for using so-called low dose IL-2 was toexploit the high IL-2 affinity of the trimeric IL-2 receptor which isconstitutively expressed on Tregs while leaving other T cells which donot express the high affinity receptor in the inactivated state.Aldesleukin (marketed as Proleukin® by Prometheus Laboratories, SanDiego, Calif.), the recombinant form of IL-2 used in these trials, isassociated with high toxicity. Aldesleukin is approved for the treatmentof metastatic melanoma and metastatic renal cancer, but its side effectsare so severe that its use is only recommended in a hospital settingwith access to intensive care (Web address:www.proleukin.com/assets/pdf/proleukin.pdf).

The clinical trials of IL-2 in autoimmune diseases have employed lowerdoses of IL-2 in order to target Treg cells, because Treg cells respondto lower concentrations of IL-2 than many other immune cell types due totheir expression of IL-2Rα (Klatzmann D, 2015 Nat Rev Immunol.15:283-94). However, even these lower doses resulted in safety andtolerability issues, and the treatments used have employed dailysubcutaneous injections, either chronically or in intermittent 5-daytreatment courses. Therefore, there is a need for an autoimmune diseasetherapy that potentiates Treg cell numbers and function, that targetsTreg cells more specifically than IL-2, that is safer and moretolerable, and that is administered less frequently.

One approach that has been suggested for improving the therapeutic indexof IL-2-based therapy for autoimmune diseases is to use variants of IL-2that are selective for Treg cells relative to other immune cells. IL-2receptors are expressed on a variety of different immune cell types,including T cells, NK cells, eosinophils, and monocytes, and this broadexpression pattern likely contributes to its pleiotropic effect on theimmune system and high systemic toxicity. In particular, activated Teffector cells express IL-2Rαβγ, as do pulmonary epithelial cells. But,activating T effector cells runs directly counter to the goal ofdown-modulating and controlling an immune response, and activatingpulmonary epithelial cells leads to known dose-limiting side effects ofIL-2 including pulmonary edema. In fact, the major side effect ofhigh-dose IL-2 immunotherapy is vascular leak syndrome (VLS), whichleads to accumulation of intravascular fluid in organs such as lungs andliver with subsequent pulmonary edema and liver cell damage. There is notreatment of VLS other than withdrawal of IL-2. Low-dose IL-2 regimenshave been tested in patients to avoid VLS, however, at the expense ofsuboptimal therapeutic results.

According to the literature, VLS is believed to be caused by the releaseof proinflammatory cytokines from IL-2-activated NK cells. However,there is strong evidence that pulmonary edema results from directbinding of IL-2 to lung endothelial cells, which expressed low tointermediate levels of functional αβγ IL-2Rs. The pulmonary edemaassociated with interaction of IL-2 with lung endothelial cells wasabrogated by blocking binding to CD25 with an anti-CD25 monoclonalantibody (mAb), in CD25-deficient host mice, or by the use ofCD122-specific IL-2/anti-IL-2 mAb (IL-2/mAb) complexes, thus preventingVLS.

Treatment with interleukin cytokines other than IL-2 has been morelimited. IL-15 displays immune cell stimulatory activity similar to thatof IL-2 but without the same inhibitory effects, thus making it apromising immunotherapeutic candidate. Clinical trials of recombinanthuman IL-15 for the treatment of metastatic malignant melanoma or renalcell cancer demonstrated appreciable changes in immune celldistribution, proliferation, and activation and suggested potentialantitumor activity (Conlon et. al., 2014). IL-15 is currently inclinical trials to treat various forms of cancer. However, IL-15 therapyis known to be associated with undesired and toxic effects, such asexacerbating certain leukemias, graft-versus-host disease, hypotension,thrombocytopenia, and liver injury. (Mishra A., et al., Cancer Cell,2012, 22(5):645-55; Alpdogan O. et al., Blood, 2005, 105(2):866-73;Conlon K C et al., J Clin Oncol, 2015, 33(1):74-82.)

The direct use of IL-2 as an agonist to bind the IL-2R and modulateimmune responses therapeutically has been problematic due itswell-documented therapeutic risks, e.g., its short serum half-life andhigh toxicity. These risks have also limited the therapeutic developmentand use of other cytokines. New forms of cytokines that reduce theserisks are needed. Disclosed herein are compositions and methodscomprising IL-2 and IL-15 and other cytokines, functional fragments andmuteins of cytokines as well as conditionally active cytokines designedto address these risks and provide needed immunomodulatory therapeutics.

The present invention is designed to address the shortcomings of directIL-2 therapy and therapy using other cytokines, for example usingcytokine blocking moieties, e.g., steric blocking polypeptides, serumhalf-life extending polypeptides, targeting polypeptides, linkingpolypeptides, including protease cleavable linkers, and combinationsthereof. Cytokines, including interleukins (e.g., IL-2, IL-7, IL-12,IL-15, IL-18, IL-21 IL-23), interferons (IFNs, including IFNalpha,IFNbeta and IFNgamma), tumor necrosis factors (e.g., TNFalpha,lymphotoxin), transforming growth factors (e.g., TGFbeta1, TGFbeta2,TGFbeta3), chemokines (C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20,CCL21), and granulocyte macrophage-colony stimulating factor (GM-CS) arehighly potent when administered to patients. As used herein, “chemokine”means a family of small cytokines with the ability to induce directedchemotaxis in nearby responsive cells Cytokines can provide powerfultherapy, but are accompanied by undesired effects that are difficult tocontrol clinically and which have limited the clinical use of cytokines.This disclosure relates to new forms of cytokines that can be used inpatients with reduced or eliminated undesired effects. In particular,this disclosure relates to pharmaceutical compositions includingchimeric polypeptides (fusion proteins), nucleic acids encoding fusionproteins and pharmaceutical formulations of the foregoing that containcytokines or active fragments or muteins of cytokines that havedecreased cytokine receptor activating activity in comparison to thecorresponding cytokine. However, under selected conditions or in aselected biological environment the chimeric polypeptides activate theircognate receptors, often with the same or higher potency as thecorresponding naturally occurring cytokine. As described herein, this istypically achieved using a cytokine blocking moiety that blocks orinhibits the receptor activating function of the cytokine, activefragment or mutein thereof under general conditions but not underselected conditions, such as those present at the desired site ofcytokine activity (e.g., an inflammatory site or a tumor).

The chimeric polypeptides and nucleic acids encoding the chimericpolypeptides can be made using any suitable method. For example, nucleicacids encoding a chimeric polypeptide can be made using recombinant DNAtechniques, synthetic chemistry or combinations of these techniques, andexpressed in a suitable expression system, such as in CHO cells.Chimeric polypeptides can similarly be made, for example by expressionof a suitable nucleic acid, using synthetic or semi-synthetic chemicaltechniques, and the like. In some embodiments, the blocking moiety canbe attached to the cytokine polypeptide via sortase-mediatedconjugation. “Sortases” are transpeptidases that modify proteins byrecognizing and cleaving a carboxyl-terminal sorting signal embedded inor terminally attached to a target protein or peptide. Sortase Acatalyzes the cleavage of the LPXTG motif (SEQ ID NO: 125) (where X isany standard amino acid) between the Thr and Gly residue on the targetprotein, with transient attachment of the Thr residue to the active siteCys residue on the enzyme, forming an enzyme-thioacyl intermediate. Tocomplete transpeptidation and create the peptide-monomer conjugate, abiomolecule with an N-terminal nucleophilic group, typically anoligoglycine motif, attacks the intermediate, displacing Sortase A andjoining the two molecules.

To form the cytokine-blocking moiety fusion protein, the cytokinepolypeptide is first tagged at the N-terminus with a polyglycinesequence, or alternatively, with at the C-terminus with a LPXTG motif(SEQ ID NO: 125). The blocking moiety or other element has respectivepeptides attached that serve as acceptor sites for the taggedpolypeptides. For conjugation to domains carrying a LPXTG acceptorpeptide (SEQ ID NO: 125) attached via its N-terminus, the polypeptidewill be tagged with an N-terminal poly-glycine stretch. For conjugationto domain carrying a poly-glycine peptide attached via its C-terminus,the polypeptide will be tagged at its C-terminus with a LPXTG sortaserecognition sequence (SEQ ID NO: 125). Recognizing poly-glycine andLPXTG (SEQ ID NO: 125) sequences, sortase will form a peptide bondbetween polymer-peptide and tagged polypeptides. The sortase reactioncleaves off glycine residues as intermediates and occurs at roomtemperature.

A variety of mechanisms can be exploited to remove or reduce theinhibition caused by the blocking moiety. For example, thepharmaceutical compositions can include an IL-2 polypeptide and ablocking moiety, e.g., a steric blocking moiety, with a proteasecleavable linker comprising a protease cleavage site located between theIL-2 polypeptide and IL-2 blocking moiety or within the IL-2 blockingmoiety. When the protease cleavage site is cleaved, the blocking moietycan dissociate from cytokine, and the cytokine can then activatecytokine receptor. A cytokine moiety can also be blocked by a specificblocking moiety, such as an antibody, which binds an epitope found onthe relevant cytokine.

Any suitable linker can be used. For example, the linker can compriseglycine-glycine, a sortase-recognition motif, or a sortase-recognitionmotif and a peptide sequence (Gly4Ser)_(n) (SEQ ID NO: 126) or(Gly₃Ser)_(n) (SEQ ID NO: 127), wherein n is 1, 2, 3, 4 or 5. Typically,the sortase-recognition motif comprises a peptide sequence LPXTG (SEQ IDNO: 125), where X is any amino acid. In some embodiments, the covalentlinkage is between a reactive lysine residue attached to the C-terminalof the cytokine polypeptide and a reactive aspartic acid attached to theN-terminal of the blocker or other domain. In other embodiments, thecovalent linkage is between a reactive aspartic acid residue attached tothe N-terminal of the cytokine polypeptide and a reactive lysine residueattached to the C-terminal of said blocker or other domain.

Accordingly, as described in detail herein, the cytokine blockingmoieties (e.g., IL-2 blocking moieties) used can be steric blockers. Asused herein, a “steric blocker” refers to a polypeptide or polypeptidemoiety that can be covalently bonded to a cytokine polypeptide directlyor indirectly through other moieties such as linkers, for example in theform of a chimeric polypeptide (fusion protein), but otherwise does notcovalently bond to the cytokine polypeptide. A steric blocker cannon-covalently bond to the cytokine polypeptide, for example thoughelectrostatic, hydrophobic, ionic or hydrogen bonding. A steric blockertypically inhibits or blocks the activity of the cytokine moiety due toits proximity to the cytokine moiety and comparative size. The stericinhibition of the cytokine moiety can be removed by spatially separatingthe cytokine moiety from the steric blocker, such as by enzymaticallycleaving a fusion protein that contains a steric blocker and a cytokinepolypeptide at a site between the steric blocker and the cytokinepolypeptide.

As described in greater detail herein, the blocking function can becombined with or due to the presence of additional functional componentsin the pharmaceutical composition, such as a targeting domain, a serumhalf-life extension element, and protease-cleavable linkingpolypeptides. For example, a serum half-life extending polypeptide canalso be a steric blocker.

Various elements ensure the delivery and activity of IL-2 preferentiallyat the site of desired IL-2 activity and to severely limit systemicexposure to the interleukin via a blocking and/or a targeting strategypreferentially linked to a serum half-life extension strategy. In thisserum half-life extension strategy, the blocked version of interleukincirculates for extended times (preferentially 1-2 or more weeks) but theactivated version has the typical serum half-life of the interleukin.

By comparison to a serum half-life extended version, the serum half-lifeof IL-2 administered intravenously is only ˜10 minutes due todistribution into the total body extracellular space, which is large,˜15 L in an average sized adult. Subsequently, IL-2 is metabolized bythe kidneys with a half-life of ˜2.5 hours. (Smith, K. “Interleukin 2immunotherapy.” Therapeutic Immunology 240 (2001)). By othermeasurements, IL-2 has a very short plasma half-life of 85 minutes forintravenous administration and 3.3 hours subcutaneous administration(Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10). In someembodiments of this invention, the half-life extension element is linkedto the interleukin via a linker which is cleaved at the site of action(e.g., by inflammation-specific or tumor-specific proteases) releasingthe interleukin's full activity at the desired site and also separatingit from the half-life extension of the uncleaved version. In suchembodiments, the fully active and free interleukin would have verydifferent pharmacokinetic (pK) properties—a half-life of hours insteadof weeks. In addition, exposure to active cytokine is limited to thesite of desired cytokine activity (e.g., an inflammatory site or tumor)and systemic exposure to active cytokine, and associated toxicity andside effects, are reduced.

Other cytokines envisioned in this invention have similar pharmacology(e.g., IL-15 as reported by Blood 2011 117:4787-4795; doi:doi.org/10.1182/blood-2010-10-311456) as IL-2 and accordingly, thedesigns of this invention address the shortcomings of using these agentsdirectly, and provide chimeric polypeptides that can have extendedhalf-life and/or be targeted to a site of desired activity (e.g., a siteof inflammation or a tumor).

If desired, IL-2 can be engineered to bind the IL-2R complex generallyor one of the three IL-2R subunits specifically with an affinity thatdiffers from that of the corresponding wild-type IL-2, for example totoselectively activate Tregs or Teff. For example, IL-2 polypeptides thatare said to have higher affinity for the trimeric form of the IL-2receptor relative to the dimeric beta/gamma form of the IL-2 receptor incomparison to wild type IL-2 can have an amino acid sequence thatincludes one of the following sets of mutations with respect to SEQ IDNO: 1 (a mature IL-2 protein comprising amino acids 21-153 of human IL-2having the Uniprot Accession No. P60568-1): (a) K64R, V69A, and Q74P;(b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A,Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A,and T133N; (g) N305, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, andS99P; (i) N305, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V69A, andQ74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R; (1) K48E, E68D, N71T,N90H, F103S, and 1114V; (m) S4P, T10A, Q11R, V69A, Q74P, N88D, andT133A; (n) E15K, N30S Y31H, K35R, K48E, V69A, Q74P, and I92T; (o) N30S,E68D, V69A, N71A, Q74P, S75P, K76R, and N90H; (p) N30S, Y31C, T37A,V69A, A73V, Q74P, H79R, and I128T; (q) N26D, N29S, N30S, K54R, E67G,V69A, Q74P, and I92T; (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P,and I92T; and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D,and I89V. This approach can also be applied to prepare muteins of othercytokines including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18,IL-23), interferons (IFNs, including IFNalpha, IFNbeta and IFNgamma),tumor necrosis factors (e.g., TNFalpha, lymphotoxin), transforminggrowth factors (e.g., TGFbeta1, TGFbeta2, TGFbeta3) and granulocytemacrophage-colony stimulating factor (GM-CS). For example, muteins canbe prepared that have desired binding affinity for a cognate receptor.

As noted above, any of the mutant IL-2 polypeptides disclosed herein caninclude the sequences described; they can also be limited to thesequences described and otherwise identical to SEQ ID NO:1. Moreover,any of the mutant IL-2 polypeptides disclosed herein can optionallyinclude a substitution of the cysteine residue at position 125 withanother residue (e.g., serine) and/or can optionally include a deletionof the alanine residue at position 1 of SEQ ID NO: 1.

Another approach to improving the therapeutic index of an IL-2 basedtherapy is to optimize the pharmacokinetics of the molecule to maximallyactivate Treg cells. Early studies of IL-2 action demonstrated that IL-2stimulation of human T cell proliferation in vitro required a minimum of5-6 hours exposure to effective concentrations of IL-2 (Cantrell, D. A.,et. al., 1984, Science, 224: 1312-1316). When administered to humanpatients, IL-2 has a very short plasma half-life of 85 minutes forintravenous administration and 3.3 hours subcutaneous administration(Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10). Becauseof its short half-life, maintaining circulating IL-2 at or above thelevel necessary to stimulate T cell proliferation for the necessaryduration necessitates high doses that result in peak IL-2 levelssignificantly above the EC50 for Treg cells or will require frequentadministration. These high IL-2 peak levels can activate IL-2Rβγreceptors and have other unintended or adverse effects, for example VLSas noted above. An IL-2 analog, or a multifunctional protein with IL-2attached to a domain that enables binding to the FcRn receptor, with alonger circulating half-life than IL-2 can achieve a target drugconcentration for a specified period of time at a lower dose than IL-2,and with lower peak levels. Such an IL-2 analog will therefore requireeither lower doses or less frequent administration than IL-2 toeffectively stimulate Treg cells. Less frequent subcutaneousadministration of an IL-2 drug will also be more tolerable for patients.A therapeutic with these characteristics will translate clinically intoimproved pharmacological efficacy, reduced toxicity, and improvedpatient compliance with therapy. Alternatively, IL-2 or muteins of IL-2(herein, “IL-2*”) can be selectively targeted to the intended site ofaction (e.g., sites of inflammation). This targeting can be achieved byone of several strategies, including the addition of domains to theadministered agent that comprise blockers of the IL-2 (or muteins) thatare cleaved away or by targeting domains or a combination of the two.

In some embodiments, IL-2* partial agonists can be tailored to bind withhigher or lower affinity depending on the desired target; for example,an IL-2* can be engineered to bind with enhanced affinity to one of thereceptor subunits and not the others. These types of partial agonists,unlike full agonists or complete antagonists, offer the ability to tunethe signaling properties to an amplitude that elicits desired functionalproperties while not meeting thresholds for undesired properties. Giventhe differential activities of the partial agonists, a repertoire ofIL-2 variants could be engineered to exhibit an even finer degree ofdistinctive signaling activities, ranging from almost full to partialagonism to complete antagonism.

In some embodiments, the IL-2* has altered affinity for IL-2Ra. In someembodiments, the IL-2* has a higher affinity for IL-2Rα than wild-typeIL-2. In other embodiments, the IL-2* has altered affinity for IL-2Rβ.In one embodiment, IL-2* has enhanced binding affinity for IL-2Rβ, e.g.,the N-terminus of IL-2Rβ, that eliminates the functional requirement forIL-2Ra. In another embodiment, an IL-2* is generated that has increasedbinding affinity for IL-2Rβ but that exhibited decreased binding toIL-2Rγ, and thereby is defective IL-2Rβγ heterodimerization andsignaling.

Blocking moieties, described in further detail below, can also be usedto favor binding to or activation of one or more receptors. In oneembodiment, blocking moieties are added such that IL-2Rβγ binding oractivation is blocked but IL-2Rα binding or activation is not changed.In another embodiment, blocking moieties are added such that IL-2Rαbinding or activation is diminished. In another embodiment, blockingmoieties are added such that binding to and or activation of all threereceptors is inhibited. This blocking may be relievable by removal ofthe blocking moieties in a particular environment, for example byproteolytic cleavage of a linker linking one or more blocking moietiesto the cytokine.

A similar approach can be applied to improve other cytokines,particularly for use as immunostimulatory agents, for example fortreating cancer. For example, in this aspect, the pharmacokineticsand/or pharmacodynamics of the cytokine (e.g., IL-2, IL-7, IL-12, IL-15,IL-18, IL-21 IL-23, IFNalpha, IFNbeta and IFNgamma, TNFalpha,lymphotoxin, TGFbeta1, TGFbeta2, TGFbeta3 GM-CSF, CXCL10, CCL19, CCL20,and CCL21 can be tailored to maximally activate effector cells (e.g.,effect T cells, NK cells) and/or cytotoxic immune response promotingcells (e.g., induce dendritic cell maturation) at a site of desiredactivity, such as in a tumor, but preferably not systemically.

Thus, provided herein are pharmaceutical compositions comprising atleast one cytokine polypeptide, such as interleukins (e.g., IL-2, IL-7,IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs, includingIFNalpha, IFNbeta and IFNgamma), tumor necrosis factors (e.g., TNFalpha,lymphotoxin), transforming growth factors (e.g., TGFbeta1, TGFbeta2,TGFbeta3), chemokines (e.g., CXCL10, CCL19, CCL20, CCL21) andgranulocyte macrophage-colony stimulating factor (GM-CS) or a functionalfragment or mutein of any of the foregoing. The polypeptide typicallyalso includes at least one linker amino acid sequence, wherein the aminoacid sequence is in certain embodiments capable of being cleaved by anendogenous protease. In one embodiment, the linker comprises an aminoacid sequence comprising HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO:128), IPVSLRSG (SEQ ID NO: 129), VPLSLYSG (SEQ ID NO: 130), orSGESPAYYTA (SEQ ID NO: 131). In other embodiments, the chimericpolypeptide further contains a blocking moiety, e.g., a steric blockingpolypeptide moiety, capable of blocking the activity of the interleukinpolypeptide. The blocking moiety, for example, can comprise a humanserum albumin (HSA) binding domain or an optionally branched ormulti-armed polyethylene glycol (PEG). Alternatively, the pharmaceuticalcomposition comprises a first cytokine polypeptide or a fragmentthereof, and blocking moiety, e.g., a steric blocking polypeptidemoiety, wherein the blocking moiety blocks the activity of the cytokinepolypeptide on the cytokine receptor, and wherein the blocking moiety incertain embodiments comprises a protease cleavable domain. In someembodiments, blockade and reduction of cytokine activity is achievedsimply by attaching additional domains with very short linkers to the Nor C terminus of the interleukin domain. In such embodiments, it isanticipated the blockade is relieved by protease digestion of theblocking moiety or of the short linker that tethers the blocker to theinterleukin. Once the domain is clipped or is released, it will nolonger be able to achieve blockade of cytokine activity.

The pharmaceutical composition e.g., chimeric polypeptide can comprisetwo or more cytokines, which can be the same cytokine polypeptide ordifferent cytokine polypeptides. For example, the two or more differenttypes of cytokines have complementary functions. In some examples, afirst cytokine is IL-2 and a second cytokine is IL-12. In someembodiments, each of the two or more different types of cytokinepolypeptides have activities that modulate the activity of the othercytokine polypeptides. In some examples of chimeric polypeptides thatcontain two cytokine polypeptides, a first cytokine polypeptide isT-cell activating, and a second cytokine polypeptide isnon-T-cell-activating. In some examples of chimeric polypeptides thatcontain two cytokine polypeptides, a first cytokine is achemoattractant, e.g., CXCL10, and a second cytokine is an immune cellactivator.

Preferably, the cytokine polypetides (including functional fragments)that are included in the fusion proteins disclosed herein are notmutated or engineered to alter the properties of the naturally occurringcytokine, including receptor binding affinity and specificity or serumhalf-life. However, changes in amino acid sequence from naturallyoccurring (including wild type) cytokine are acceptable to facilitatecloning and to achieve desired expression levels, for example.

CD25 Binding

CD25 binding is often discouraged in modified IL-2 constructs. Incontrast, the IL-2 polypeptides described herein preferably are notmodified to avoid CD25 binding. Preferably, the IL-2 polypeptidesdescribed herein bind CD25. Typically, the IL-2 fusion proteinsdescribed herein are capable of CD25 binding and blocking is directed tointeractions with IL-2R beta and gamma (CD122 and CD132).

Blocking Moiety

The blocking moiety can be any moiety that inhibits the ability of thecytokine to bind and/or activate its receptor. The blocking moiety caninhibit the ability of the cytokine to bind and/or activate its receptorsterically blocking and/or by noncovalently binding to the cytokine.Examples of suitable blocking moieties include the full length or acytokine-binding fragment or mutein of the cognate receptor of thecytokine. Antibodies and fragments thereof including, a polyclonalantibody, a recombinant antibody, a human antibody, a humanized antibodya single chain variable fragment (scFv), single-domain antibody such asa heavy chain variable domain (VH), a light chain variable domain (VL)and a variable domain of camelid-type nanobody (VHH), a dAb and the likethat bind the cytokine can also be used. Other suitable antigen-bindingdomain that bind the cytokine can also be used, includenon-immunoglobulin proteins that mimic antibody binding and/or structuresuch as, anticalins, affilins, affibody molecules, affimers, affitins,alphabodies, avimers, DARPins, fynomers, kunitz domain peptides,monobodies, and binding domains based on other engineered scaffolds suchas SpA, GroEL, fibronectin, lipocallin and CTLA4 scaffolds. Furtherexamples of suitable blocking polypeptides include polypeptides thatsterically inhibit or block binding of the cytokine to its cognatereceptor. Advantageously, such moieties can also function as half-lifeextending elements. For example, a peptide that is modified byconjugation to a water-soluble polymer, such as PEG, can stericallyinhibit or prevent binding of the cytokine to its receptor.Polypeptides, or fragments thereof, that have long serum half-lives canalso be used, such as serum albumin (human serum albumin),immunoglobulin Fc, transferrin and the like, as well as fragments andmuteins of such polypeptides.

Antibodies and antigen-binding domains that bind to, for example, aprotein with a long serum half-life such as HSA, immunoglobulin ortransferrin, or to a receptor that is recycled to the plasma membrane,such as FcRn or transferrin receptor, can also inhibit the cytokine,particularly when bound to their antigen. Examples of suchantigen-binding polypeptides include a single chain variable fragment(scFv), single-domain antibody such as a heavy chain variable domain(VH), a light chain variable domain (VL) and a variable domain ofcamelid-type nanobody (VHH), a dAb and the like. Other suitableantigen-binding domain that bind the cytokine can also be used, includenon-immunoglobulin proteins that mimic antibody binding and/or structuresuch as, anticalins, affilins, affibody molecules, affimers, affitins,alphabodies, avimers, DARPins, fynomers, kunitz domain peptides,monobodies, and binding domains based on other engineered scaffolds suchas SpA, GroEL, fibronectin, lipocallin and CTLA4 scaffolds.

In illustrative examples, when IL-2 is the cytokine in the chimericpolypeptide, the blocking moiety can be the full length or fragment ormutein of the alpha chain of IL-2 receptor (IL-2Ra) or beta (IL-2Rβ) orgamma chain of IL-2 receptor (IL-2Rγ), an anti-IL-2 single-domainantibody (dAb) or scFv, a Fab, an anti-CD25 antibody or fragmentthereof, and anti-HAS dAb or scFv, and the like.

Additional Aspects of the Invention

-   -   1. A fusion protein comprising a cytokine moiety that is        operably linked to a binding moiety, the binding moiety        comprising a non-CDR loop and a cleavable linker, wherein the        binding moiety is capable of masking the binding the cytokine to        its receptor and/or the activation of the receptor by the        cytokine.    -   2. The fusion protein of aspect 1, wherein the binding moiety is        a natural peptide, a synthetic peptide, an engineered scaffold,        or an engineered bulk serum protein.    -   3. The fusion protein of aspect 1 or 2, wherein the engineered        scaffold comprises a sdAb, a scFv, a Fab, a VHH, a fibronectin        type III domain, immunoglobulin-like scaffold, DARPin, cystine        knot peptide, lipocalin, three-helix bundle scaffold, protein        G-related albumin-binding module, or a DNA or RNA aptamer        scaffold.    -   4. The fusion protein of any one of aspects 1-2, wherein the        binding moiety is capable of binding to a bulk serum protein.    -   5. The fusion protein of any one of aspects 1-3, wherein the        non-CDR loop is from a variable domain, a constant domain, a        C1-set domain, a C2-set domain, an I-domain, or any combinations        thereof.    -   6. The fusion protein of any one of aspects 1-4, wherein the        binding moiety further comprises complementarity determining        regions (CDRs).    -   7. The fusion protein of aspect 5, wherein the binding moiety is        capable of binding to the bulk serum protein.    -   8. The fusion protein of aspect 6, wherein the bulk serum        protein is a half-life extending protein.    -   9. The fusion protein of aspect 6 or 7, wherein the bulk serum        protein is albumin, transferrin, Factor XIII, or Fibrinogen.    -   10. The fusion protein of any one of aspects 5-8, wherein the        CDR loop provides the binding site specific for the bulk serum        protein or the immunoglobulin light chain, or any combinations        thereof.    -   11. The fusion protein of any one of aspects 1-9, wherein the        cleavable linker comprises a cleavage site.    -   12. The fusion protein of aspect 10, wherein the cleavage site        is recognized by a protease.    -   13. The fusion protein of aspect 11, wherein the binding moiety        is bound to the cytokine.    -   14. The fusion protein of aspect 11 or 13, wherein the binding        moiety is covalently linked to the cytokine.    -   15. The fusion protein of aspect 11, 13, or 14, wherein the        binding moiety is capable of masking the binding of the cytokine        to its target via specific intermolecular interactions between        the binding moiety and the cytokine.    -   16. The fusion protein of any one of aspects 11-14, wherein the        non-CDR loop provides a binding site specific for binding of the        moiety to the cytokine.    -   17. The fusion protein of any one of aspects 11-15, wherein upon        cleavage of the cleavable linker, the binding moiety is        separated from the cytokine and the cytokine binds to its        target.    -   18. The fusion protein of any one of aspects 1-16, wherein the        cytokine binds to a cytokine receptor.    -   19. The fusion protein of aspect 17, wherein the cytokine        receptor comprises a type I cytokine receptor, a type I IL        receptor, a type II IL receptor, a chemokine receptor, or a        tumor necrosis receptor superfamily receptor.    -   20. The fusion protein of any one of aspects 1-18, wherein the        cleavable linker comprises a cleavage site.    -   21. The fusion protein of aspect 20, wherein the cleavage site        is recognized by a protease.    -   22. The fusion protein of aspect 21, wherein the protease        cleavage site is recognized by a serine protease, a cysteine        protease, an aspartate protease, a threonine protease, a        glutamic acid protease, a metalloproteinase, a gelatinase, or a        asparagine peptide lyase.    -   23. The fusion protein of aspect 21, wherein the protease        cleavage site is recognized by a Cathepsin B, a Cathepsin C, a        Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a        kallikrein, a hK1, a hK10, a hK15, a plasmin, a collagenase, a        Type IV collagenase, a stromelysin, a Factor Xa, a        chymotrypsin-like protease, a trypsin-like protease, a        elastase-like protease, a subtilisin-like protease, an        actinidain, a bromelain, a calpain, a caspase, a caspase-3, a        Mir1-CP, a papain, a HIV-1 protease, a HSV protease, a CMV        protease, a chymosin, a renin, a pepsin, a matriptase, a        legumain, a plasmepsin, a nepenthesin, a metalloexopeptidase, a        metalloendopeptidase, a matrix metalloprotease (MMP), a MMP1, a        MMP2, a MMP3, a MMP8, a MMP9, a MMP10, a MMP11, a MMP12, a        MMP13, a MMP14, an ADAM10, an ADAM17, an ADAM12, an urokinase        plasminogen activator (uPA), an enterokinase, a        prostate-specific target (PSA, hK3), an interleukin-1β        converting enzyme, a thrombin, a FAP (FAP-α), a dipeptidyl        peptidase, or dipeptidyl peptidase IV (DPPIV/CD26), a type II        transmembrane serine protease (TTSP), a neutrophil elastase, a        cathepsin G, a proteinase 3, a neutrophil serine protease 4, a        mast cell chymase, a mast cell tryptase, a dipeptidyl peptidase,        and a dipeptidyl peptidase IV (DPPIV/CD26).    -   24. A conditionally active binding protein comprising a binding        moiety (M) which comprises a non-CDR loop, a cytokine, and a        cleavable linker (L), wherein the non-CDR loop is capable of        binding to the cytokine, and wherein the binding moiety is        capable of inhibiting the binding of the cytokine to its        receptor and/or inhibiting activation of the receptor by the        cytokine.    -   25. The conditionally active binding protein of aspect 24,        wherein the binding moiety is capable of binding to a half-life        extending protein.    -   26. The conditionally active binding protein of aspect 24 or 25,        wherein the binding moiety is a natural peptide, a synthetic        peptide, an engineered scaffold, or an engineered serum bulk        protein.    -   27. The conditionally active binding protein of aspect 26,        wherein the engineered scaffold comprises a sdAb, a scFv, a Fab,        a VHH, a fibronectin type III domain, immunoglobulin-like        scaffold, DARPin, cystine knot peptide, lipocalin, three-helix        bundle scaffold, protein G-related albumin-binding module, or a        DNA or RNA aptamer scaffold.    -   28. The conditionally active binding protein of any one of        aspects 24-27, wherein the non-CDR-loop is from a variable        domain, a constant domain, a C1-set domain, a C2-set domain, an        I-domain, or any combinations thereof.    -   29. The conditionally active binding protein of any one of        aspects 24-28, wherein the binding moiety further comprises        complementarity determining regions (CDRs).    -   30. The conditionally active binding protein of any one of        aspects 24-29, wherein the binding moiety comprises a binding        site specific for a bulk serum protein.    -   31. The conditionally active binding protein of aspect 30,        wherein the bulk serum protein is albumin, transferrin, Factor        XIII, or Fibrinogen.    -   32. The conditionally active binding protein of any one of        aspects 29-31, wherein the CDRs provide the binding site        specific for the bulk serum protein or the immunoglobulin light        chain, or any combinations thereof.    -   33. The conditionally active binding protein of any one of        aspects 29-32, wherein the binding moiety is capable of masking        the binding of the cytokine to its target via specific        intermolecular interactions between the binding moiety and the        cytokine.    -   34. The conditionally active binding protein of any one of        aspects 29-33, wherein the non-CDR loop provides a binding site        specific for binding of the binding moiety to the cytokine.    -   35. The conditionally active binding protein of any one of        aspects 24-34, wherein the cytokine binds to a cytokine        receptor.    -   36. The conditionally active binding protein of aspect 35,        wherein the cytokine receptor comprises a type I cytokine        receptor, a type I IL receptor, a type II IL receptor, a        chemokine receptor, or a tumor necrosis receptor superfamily        receptor.    -   37. The conditionally active binding protein of aspect 24-36,        wherein the cleavable linker comprises a cleavage site.    -   38. The conditionally active binding protein of aspect 37,        wherein the cleavage site is recognized by a protease.    -   39. The conditionally active binding protein of aspect 38,        wherein the protease cleavage site is recognized by a serine        protease, a cysteine protease, an aspartate protease, a        threonine protease, a glutamic acid protease, a        metalloproteinase, a gelatinase, or a asparagine peptide lyase.    -   40. The conditionally active binding protein of aspect 38,        wherein the protease cleavage site is recognized by a Cathepsin        B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a        Cathepsin L, a kallikrein, a hK1, a hK10, a hK15, a plasmin, a        collagenase, a Type IV collagenase, a stromelysin, a Factor Xa,        a chymotrypsin-like protease, a trypsin-like protease, a        elastase-like protease, a subtilisin-like protease, an        actinidain, a bromelain, a calpain, a caspase, a caspase-3, a        Mir1-CP, a papain, a HIV-1 protease, a HSV protease, a CMV        protease, a chymosin, a renin, a pepsin, a matriptase, a        legumain, a plasmepsin, a nepenthesin, a metalloexopeptidase, a        metalloendopeptidase, a matrix metalloprotease (MMP), a MMP1, a        MMP2, a MMP3, a MMP8, a MMP9, a MMP10, a MMP11, a MMP12, a        MMP13, a MMP14, an ADAM10, an ADAM17, an ADAM12, an urokinase        plasminogen activator (uPA), an enterokinase, a        prostate-specific target (PSA, hK3), an interleukin-1β        converting enzyme, a thrombin, a FAP (FAP-α), a dipeptidyl        peptidase, or dipeptidyl peptidase IV (DPPIV/CD26), a type II        transmembrane serine protease (TTSP), a neutrophil elastase, a        cathepsin G, a proteinase 3, a neutrophil serine protease 4, a        mast cell chymase, a mast cell tryptase, a dipeptidyl peptidase,        and a dipeptidyl peptidase IV (DPPIV/CD26).    -   41. The conditionally active binding protein of aspect 24,        further comprising a half-life extension domain bound to the        binding moiety, wherein the half-life extension domain provides        the binding protein with a safety switch, and wherein upon        cleavage of the linker the binding protein is activated by        separation of the binding moiety and the half-life extension        domain from the cytokine, and the binding protein is thereby        separated from the safety switch.    -   42. The conditionally active binding protein of aspect 41,        wherein the cleavage of the linker is in a tumor        microenvironment.    -   43. A conditionally active binding protein, comprising a binding        moiety that binds a cytokine via a non-CDR loop within the        binding moiety, wherein the binding moiety is further linked to        a half-life extension domain and comprises a cleavable linker,        wherein the binding protein has an extended half-life prior to        its activation by cleavage of the linker, and wherein upon        activation the binding moiety and the half-life extension domain        are separated from the cytokine, and wherein the binding        protein, in its activated state, does not have an extended        half-life.    -   44. The conditionally active binding protein of aspect 43,        wherein the cleavage of the linker is in a tumor        microenvironment.        In Vivo Half-Life Extension Elements

Preferably, the chimeric polypeptides comprise an in vivo half-lifeextension element. Increasing the in vivo half-life of therapeuticmolecules with naturally short half-lives allows for a more acceptableand manageable dosing regimen without sacrificing effectiveness. As usedherein, a “half-life extension element” is a part of the chimericpolypeptide that increases the in vivo half-life and improve pK, forexample, by altering its size (e.g., to be above the kidney filtrationcutoff), shape, hydrodynamic radius, charge, or parameters ofabsorption, biodistribution, metabolism, and elimination. An exemplaryway to improve the pK of a polypeptide is by expression of an element inthe polypeptide chain that binds to receptors that are recycled to theplasma membrane of cells rather than degraded in the lysosomes, such asthe FcRn receptor on endothelial cells and transferrin receptor. Threetypes of proteins, e.g., human IgGs, HSA (or fragments), andtransferrin, persist for much longer in human serum than would bepredicted just by their size, which is a function of their ability tobind to receptors that are recycled rather than degraded in thelysosome. These proteins, or fragments of them that retain the FcRnbinding are routinely linked to other polypeptides to extend their serumhalf-life. In one embodiment, the half-life extension element is a humanserum albumin (HSA) binding domain. HSA (SEQ ID NO: 2) may also bedirectly bound to the pharmaceutical compositions or bound via a shortlinker Fragments of HSA may also be used. HSA and fragments thereof canfunction as both a blocking moiety and a half-life extension element.Human IgGs and Fc fragments can also carry out a similar function.

The serum half-life extension element can also be antigen-bindingpolypeptide that binds to a protein with a long serum half-life such asserum albumin, transferrin and the like. Examples of such polypeptidesinclude antibodies and fragments thereof including, a polyclonalantibody, a recombinant antibody, a human antibody, a humanized antibodya single chain variable fragment (scFv), single-domain antibody such asa heavy chain variable domain (VH), a light chain variable domain (VL)and a variable domain of camelid-type nanobody (VHH), a dAb and thelike. Other suitable antigen-binding domain include non-immunoglobulinproteins that mimic antibody binding and/or structure such as,anticalins, affilins, affibody molecules, affimers, affitins,alphabodies, avimers, DARPins, fynomers, kunitz domain peptides,monobodies, and binding domains based on other engineered scaffolds suchas SpA, GroEL, fibronectin, lipocallin and CTLA4 scaffolds. Furtherexamples of antigen-binding polypeptides include a ligand for a desiredreceptor, a ligand-binding portion of a receptor, a lectin, and peptidesthat binds to or associates with one or more target antigens.

Some preferred serum half-life extension elements are polypeptides thatcomprise complementarity determining regions (CDRs), and optionallynon-CDR loops. Advantageously, such serum half-life extension elementscan extend the serum half-life of the cytokine, and also function asinhibitors of the cytokine (e.g., via steric blocking, non-covalentinteraction or combination thereof) and/or as targeting domains. In someinstances, the serum half-life extension elements are domains derivedfrom an immunoglobulin molecule (Ig molecule) or engineered proteinscaffolds that mimic antibody structure and/or binding activity. The Igmay be of any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgMetc). A polypeptide chain of an Ig molecule folds into a series ofparallel beta strands linked by loops. In the variable region, three ofthe loops constitute the “complementarity determining regions” (CDRs)which determine the antigen binding specificity of the molecule. An IgGmolecule comprises at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen bindingfragment thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs) withare hypervariable in sequence and/or involved in antigen recognitionand/or usually form structurally defined loops, interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In some embodiments of this disclosure, atleast some or all of the amino acid sequences of FR1, FR2, FR3, and FR4are part of the “non-CDR loop” of the binding moieties described herein.As shown in FIG. 5, a variable domain of an immunoglobulin molecule hasseveral beta strands that are arranged in two sheets. The variabledomains of both light and heavy immunoglobulin chains contain threehypervariable loops, or complementarity-determining regions (CDRs). Thethree CDRs of a V domain (CDR1, CDR2, CDR3) cluster at one end of thebeta barrel. The CDRs are the loops that connect beta strands B-C,C′-C″, and F-G of the immunoglobulin fold, whereas the bottom loops thatconnect beta strands AB, CC′, C″-D and E-F of the immunoglobulin fold,and the top loop that connects the D-E strands of the immunoglobulinfold are the non-CDR loops. In some embodiments of this disclosure, atleast some amino acid residues of a constant domain, CH1, CH2, or CH3,are part of the “non-CDR loop” of the binding moieties described herein.Non-CDR loops comprise, in some embodiments, one or more of AB, CD, EF,and DE loops of a C1-set domain of an Ig or an Ig-like molecule; AB,CC′, EF, FG, BC, and EC′ loops of a C2-set domain of an Ig or an Ig-likemolecule; DE, BD, GF, A(A1A2)B, and EF loops of I(Intermediate)-setdomain of an Ig or Ig-like molecule.

Within the variable domain, the CDRs are believed to be responsible forantigen recognition and binding, while the FR residues are considered ascaffold for the CDRs. However, in certain cases, some of the FRresidues play an important role in antigen recognition and binding.Framework region residues that affect Ag binding are divided into twocategories. The first are FR residues that contact the antigen, thus arepart of the binding-site, and some of these residues are close insequence to the CDRs. Other residues are those that are far from theCDRs in sequence, but are in close proximity to it in the 3-D structureof the molecule, e.g., a loop in heavy chain. The serum half-lifeextension domain (e.g., a domain that comprises CDRs) can comprise atleast one non-CDR loop. In some embodiments, a non-CDR loop provides abinding site for binding to a cytokine, bulk serum protein or othertarget antigen.

The serum half-life extension element, in addition to or alternativelyto containing CDRs, comprises a non-CDR loop. In some embodiments, thenon-CDR loop is modified to generate an antigen binding site specificfor a desired target antigen, such as a bulk serum protein, such asalbumin, or for the cytokine moiety or other targeting antigen. It iscontemplated that various techniques can be used for modifying thenon-CDR loop, e.g., site-directed mutagenesis, random mutagenesis,insertion of at least one amino acid that is foreign to the non-CDR loopamino acid sequence, amino acid substitution. An antigen peptide isinserted into a non-CDR loop, in some examples. In some examples, anantigenic peptide is substituted for the non-CDR loop. The modification,to generate an antigen binding site, is in some cases in only onenon-CDR loop. In other instances, more than one non-CDR loop aremodified. For instance, the modification is in any one of the non-CDRloops shown in FIG. 5, i.e., AB, CC′, C″ D, EF, and D-E. In some cases,the modification is in the DE loop. In other cases the modifications arein all four of AB, CC′, C″-D, E-F loops.

In some examples, the serum half-life extension element has dual bindingspecificity and contains CDRs that specifically bind a bulk serumproteins, such as serum albumin, and non-CDR loops that specificallybind and block the cytokine domain. In other examples, the serumhalf-life extension element contains CDRs that specifically bind atarget antigen, such as the cytokine domain or other target antigen, andnon-CDR loops that specifically bind a bulk serum protein, such as serumalbumin. Preferably, the serum half-life extension element inhibitsbinding of the cytokine domain to the cognate cytokine receptor, e.g.,via steric occlusion, via specific intermolecular interactions, or acombination of both.

In some embodiments, the serum half-life extension element noncovalentlybinds directly to the cytokine and inhibit its activity.

In certain examples, the binding moiety binds to a cytokine via one ormore of AB, CC′, C″ D, and E-F loop and binds to a bulk-serum protein,such as albumin, via one or more of BC, C′C″, and FG loop. In certainexamples, the binding moiety binds to a bulk serum protein, such asalbumin, via its AB, CC′, C″ D, or EF loop and binds to a cytokine viaits BC, C′C″, or FG loop. In certain examples, the binding moiety of thebinds to a bulk serum protein, such as albumin, via its AB, CC′, C″ D,and EF loop and is bound to a cytokine via its BC, C′C″, and FG loop. Incertain examples, the binding moiety binds to a bulk serum protein, suchas albumin, via one or more of AB, CC′, C″ D, and E-F loop and binds toa cytokine, via one or more of BC, C′C″, and FG loop.

The binding moieties are any kinds of polypeptides. For example, incertain instances the binding moieties are natural peptides, syntheticpeptides, or fibronectin scaffolds, or engineered bulk serum proteins.The bulk serum protein comprises, for example, albumin, fibrinogen, or aglobulin. In some embodiments, the binding moieties are an engineeredscaffolds. Engineered scaffolds comprise, for example, sdAb, a scFv, aFab, a VHH, a fibronectin type III domain, immunoglobulin-like scaffold(as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin,cystine knot peptide, lipocalin, three-helix bundle scaffold, proteinG-related albumin-binding module, or a DNA or RNA aptamer scaffold.

In some cases, the serum half-life extension element binds to thecytokine domain via its non-CDR loops and the cytokine domain is furtherconnected to a targeting domain as described herein. In some cases, theserum half-life extending element comprises a binding site for a bulkserum protein. In some embodiments, the CDRs provide the binding sitefor the bulk serum protein. The bulk serum protein is, in some examples,a globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer,Factor XIII, Fibrinogen, IgE, or pentameric IgM. In some embodiments,the CDR form a binding site for an immunoglobulin light chain, such asan Igκ free light chain or an Igλ free light chain.

One exemplary conditionally active protein is shown in FIG. 6. In theillustrated example, the non-CDR loops in a serum albumin binding domain(e.g., a dAb) can form a binding site for the cytokine IL-2. In thisexample, the binding site for serum albumin can be formed by the CDRs ofthe serum albumin binding domain.

The serum half-life extension element can be any type of binding domain,including but not limited to, domains from a monoclonal antibody, apolyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody. In some embodiments, the binding moiety is a singlechain variable fragment (scFv), single-domain antibody such as a heavychain variable domain (VH), a light chain variable domain (VL) and avariable domain (VHH) of camelid derived nanobody. In other embodiments,the binding moieties are non-Ig binding domains, i.e., antibody mimetic,such as anticalins, affilins, affibody molecules, affimers, affitins,alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, andmonobodies.

In other embodiments, the serum half-life extension element can be awater-soluble polymer or a peptide that is conjugated to a water-solublepolymer, such as PEG. “PEG,” “polyethylene glycol” and “poly(ethyleneglycol)” as used herein, are interchangeable and encompass anynonpeptidic water-soluble poly(ethylene oxide). The term “PEG” alsomeans a polymer that contains a majority, that is to say, greater than50%, of —OCH₂CH₂—repeating subunits. With respect to specific forms, thePEG can take any number of a variety of molecular weights, as well asstructures or geometries such as “branched,” “linear,” “forked,”“multifunctional,” and the like, to be described in greater detailbelow. The PEG is not limited to a particular structure and can belinear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG),branched or multi-armed (e.g., forked PEG or PEG attached to a polyolcore), a dendritic (or star) architecture, each with or without one ormore degradable linkages. Moreover, the internal structure of the PEGcan be organized in any number of different repeat patterns and can beselected from the group consisting of homopolymer, alternatingcopolymer, random copolymer, block copolymer, alternating tripolymer,random tripolymer, and block tripolymer. PEGs can be conjugated topolypeptide and peptides through any suitable method. Typically areactive PEG derivative, such as N-hydroxysuccinamidyl ester PEG, isreacted with a peptide or polypeptide that includes amino acids with aside chain that contains an amine, sulfhydryl, carboxylic acid orhydroxyl functional group, such as cysteine, lysine, asparagine,glutamine, theonine, tyrosine, serine, aspartic acid, and glutamic acid.

Targeting and Retention Domains

For certain applications, it may be desirable to maximize the amount oftime the construct is present in its desired location in the body. Thiscan be achieved by including one further domain in the chimericpolypeptide (fusion protein) to influence its movements within the body.For example, the chimeric nucleic acids can encode a domain that directsthe polypeptide to a location in the body, e.g., tumor cells or a siteof inflammation; this domain is termed a “targeting domain” and/orencode a domain that retains the polypeptide in a location in the body,e.g., tumor cells or a site of inflammation; this domain is termed a“retention domain”. In some embodiments a domain can function as both atargeting and a retention domain. In some embodiments, the targetingdomain and/or retention domain are specific to a protease-richenvironment. In some embodiments, the encoded targeting domain and/orretention domain are specific for regulatory T cells (Tregs), forexample targeting the CCR4 or CD39 receptors. Other suitable targetingand/or retention domains comprise those that have a cognate ligand thatis overexpressed in inflamed tissues, e.g., the IL-1 receptor, or theIL-6 receptor. In other embodiments, the suitable targeting and/orretention domains comprise those who have a cognate ligand that isoverexpressed in tumor tissue, e.g., Epcam, CEA or mesothelin. In someembodiments, the targeting domain is linked to the interleukin via alinker which is cleaved at the site of action (e.g., by inflammation orcancer specific proteases) releasing the interleukin full activity atthe desired site. In some embodiments, the targeting and/or retentiondomain is linked to the interleukin via a linker which is not cleaved atthe site of action (e.g., by inflammation or cancer specific proteases),causing the cytokine to remain at the desired site.

Antigens of choice, in some cases, are expressed on the surface of adiseased cell or tissue, for example a tumor or a cancer cell. Antigensuseful for tumor targeting and retention include but are not limited toEpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. Pharmaceuticalcompositions disclosed herein, also include proteins comprising twotargeting and/or retention domains that bind to two different targetantigens known to be expressed on a diseased cell or tissue. Exemplarypairs of antigen binding domains include but are not limited toEGFR/CEA, EpCAM/CEA, and HER-2/HER-3.

Suitable targeting and/or retention domains include antigen-bindingdomains, such as antibodies and fragments thereof including, apolyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody a single chain variable fragment (scFv),single-domain antibody such as a heavy chain variable domain (VH), alight chain variable domain (VL) and a variable domain of camelid-typenanobody (VHH), a dAb and the like. Other suitable antigen-bindingdomain include non-immunoglobulin proteins that mimic antibody bindingand/or structure such as, anticalins, affilins, affibody molecules,affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitzdomain peptides, monobodies, and binding domains based on otherengineered scaffolds such as SpA, GroEL, fibronectin, lipocallin andCTLA4 scaffolds. Further examples of antigen-binding polypeptidesinclude a ligand for a desired receptor, a ligand-binding portion of areceptor, a lectin, and peptides that binds to or associates with one ormore target antigens.

In some embodiments, the targeting and/or retention domains specificallybind to a cell surface molecule. In some embodiments, the targetingand/or retention domains specifically bind to a tumor antigen. In someembodiments, the targeting polypeptides specifically and independentlybind to a tumor antigen selected from at least one of Fibroblastactivation protein alpha (FAPa), Trophoblast glycoprotein (5T4),Tumor-associated calcium signal transducer 2 (Trop2), Fibronectin EDB(EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3,cMet, CEA, and FOLR1. In some embodiments, the targeting polypeptidesspecifically and independently bind to two different antigens, whereinat least one of the antigens is a tumor antigen selected from Fibroblastactivation protein alpha (FAPa), Trophoblast glycoprotein (5T4),Tumor-associated calcium signal transducer 2 (Trop2), Fibronectin EDB(EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3,cMet, CEA, and FOLR1.

The targeting and/or retention antigen can be a tumor antigen expressedon a tumor cell. Tumor antigens are well known in the art and include,for example, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA,and CEA. 5T4, AFP, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166,CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56,CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33,FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE,mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR,PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, WT1.

The targeting and/or retention antigen can be an immune checkpointprotein. Examples of immune checkpoint proteins include but are notlimited to CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT,TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1,CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR,LAG-3, TIM-3, or VISTA.

The targeting and/or retention antigen can be a cell surface moleculesuch as a protein, lipid or polysaccharide. In some embodiments, atargeting and/or retention antigen is a on a tumor cell, virallyinfected cell, bacterially infected cell, damaged red blood cell,arterial plaque cell, inflamed or fibrotic tissue cell. The targetingand/or retention antigen can comprise an immune response modulator.Examples of immune response modulator include but are not limited togranulocyte-macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), granulocyte colony stimulating factor(G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12(IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3,or GITR.

The targeting and/or retention antigen can be a cytokine receptor.Examples, of cytokine receptors include but are not limited to Type Icytokine receptors, such as GM-CSF receptor, G-CSF receptor, Type I ILreceptors, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; TypeII Cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2),IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), Type II ILreceptors; chemokine receptors, such as CC chemokine receptors, CXCchemokine receptors, CX3C chemokine receptors, XC chemokine receptors;tumor necrosis receptor superfamily receptors, such as TNFRSF5/CD40,TNFRSF8/CD30, TNFRSF7/CD27, TNFRSF1A/TNFR1/CD120a,TNFRSF1B/TNFR2/CD120b; TGF-beta receptors, such as TGF-beta receptor 1,TGF-beta receptor 2; Ig super family receptors, such as IL-1 receptors,CSF-1R, PDGFR (PDGFRA, PDGFRB), SCFR.

Linkers

As stated above, the pharmaceutical compositions comprise one or morelinker sequences. A linker sequence serves to provide flexibilitybetween polypeptides, such that, for example, the blocking moiety iscapable of inhibiting the activity of the cytokine polypeptide. Thelinker sequence can be located between any or all of the cytokinepolypeptide, the serum half-life extension element, and/or the blockingmoiety. As described herein at least one of the linkers is proteasecleavable, and contains a (one or more) cleavage site for a (one ormore) desired protease. Preferably, the desired protease is enriched orselectively expressed at the desired site of cytokine activity (e.g.,the tumor microenvironment). Thus, the fusion protein is preferentiallyor selectively cleaved at the site of desired cytokine activity.

Suitable linkers can be of different lengths, such as from 1 amino acid(e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids,from 3 amino acids to 12 amino acids, including 4 amino acids to 10amino acids, amino acids to 9 amino acids, 6 amino acids to 8 aminoacids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60amino acids.

The orientation of the components of the pharmaceutical composition, arelargely a matter of design choice and it is recognized that multipleorientations are possible and all are intended to be encompassed by thisdisclosure. For example, a blocking moiety can be located C-terminallyor N-terminally to a cytokine polypeptide.

Proteases known to be associated with diseased cells or tissues includebut are not limited to serine proteases, cysteine proteases, aspartateproteases, threonine proteases, glutamic acid proteases,metalloproteases, asparagine peptide lyases, serum proteases,cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E,Cathepsin K, Cathepsin L, kallikreins, hK1, hK10, hK15, plasmin,collagenase, Type IV collagenase, stromelysin, Factor Xa,chymotrypsin-like protease, trypsin-like protease, elastase-likeprotease, subtili sin-like protease, actinidain, bromelain, calpain,caspases, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMVprotease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin,nepenthesin, metalloexopeptidases, metalloendopeptidases, matrixmetalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11,MMP14, urokinase plasminogen activator (uPA), enterokinase,prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme,thrombin, FAP (FAP-a), dipeptidyl peptidase, meprins, granzymes anddipeptidyl peptidase IV (DPPIV/CD26). Proteases capable of cleavingamino acid sequences encoded by the chimeric nucleic acid sequencesprovided herein can, for example, be selected from the group consistingof a prostate specific antigen (PSA), a matrix metalloproteinase (MMP),an A Disintigrin and a Metalloproteinase (ADAM), a plasminogenactivator, a cathepsin, a caspase, a tumor cell surface protease, and anelastase. The MMP can, for example, be matrix metalloproteinase 2 (MMP2)or matrix metalloproteinase 9 (MMP9).

Proteases useful in the methods disclosed herein are presented in Table1, and exemplary proteases and their cleavage site are presented inTable 1a:

TABLE 1 Proteases relevant to inflammation and cancer ProteaseSpecificity Other aspects Secreted by killer T cells: Granzyme B (grB)Cleaves after Asp Type of serine protease; strongly residues (asp-ase)implicated in inducing perforin-dependent target cell apoptosis GranzymeA (grA) trypsin-like, cleaves after Type of serine protease; basicresidues Granzyme H (grH) Unknown substrate Type of serine protease;specificity Other granzymes are also secreted by killer T cells, but notall are present in humans Caspase-8 Cleaves after Asp Type of cysteineprotease; plays essential residues role in TCR-induced cellularexpansion- exact molecular role unclear Mucosa-associated Cleaves afterarginine Type of cysteine protease; likely acts both lymphoid tissueresidues as a scaffold and proteolytically active (MALT1) enzyme in theCBM-dependent signaling pathway Tryptase Targets: angiotensin I, Type ofmast cell-specific serine protease; fibrinogen, prourokinase,trypsin-like; resistant to inhibition by TGFβ; preferentiallymacromolecular protease inhibitors cleaves proteins after expressed inmammals due to their lysine or arginine tetrameric structure, with allsites facing residues narrow central pore; also associated withinflammation Associated with inflammation: Thrombin Targets: FGF-2, Typeof serine protease; modulates HB-EGF, Osteo-pontin, activity of vasculargrowth factors, PDGF, VEGF chemokines and extracellular proteins;strengthens VEGF-induced proliferation; induces cell migration;angiogenic factor; regulates hemostasis Chymase Exhibit chymotrypsin-Type of mast cell-specific serine protease like specificity, cleavingproteins after aromatic amino acid residues Carboxypeptidase A Cleavesamino acid Type of zinc-dependent metalloproteinase (MC-CPA) residuesfrom C-terminal end of peptides and proteins Kallikreins Targets: highmolecular Type of serine protease; modulate weight relaxation response;contribute to kininogen, pro-urokinase inflammatory response; fibrindegradation Elastase Targets: E-cadherin, GM- Type of neutrophil serineprotease; CSF, IL-1, IL-2, IL-6, degrades ECM components; regulates IL8,p38^(MAPK), TNFα, VE- inflammatory response; activates pro- cadherinapoptotic signaling Cathepsin G Targets: EGF, ENA-78, Type of serineprotease; degrades ECM IL-8, MCP-1, MMP-2, components; chemo-attractantof MT1-MMP, leukocytes; regulates inflammatory PAI-1, RANTES, TGFβ,response; promotes apoptosis TNFα PR-3 Targets: ENA-78, IL-8, Type ofserine protease; promotes IL-18, JNK, p38^(MAPK), inflammatory response;activates pro- TNFα apoptotic signaling Granzyme M (grM) Cleaves afterMet and Type of serine protease; only expressed in other long,unbranched NK cells hydrophobic residues Calpains Cleave between Arg andFamily of cysteine proteases; calcium- Gly dependent; activation isinvolved in the process of numerous inflammation- associated diseases

TABLE 1a Exemplary Proteases and Protease Recognition Sequences ProteaseCleavage Domain Sequence SEQ ID NO: MMP7 KRALGLPG  3 MMP7(DE)₈RPLALWRS(DR)₈  4 MMP9 PR(S/T)(L/I)(S/T)  5 MMP9 LEATA  6 MMP11GGAANLVRGG  7 MMP14 SGRIGFLRTA  8 MMP PLGLAG  9 MMP PLGLAX 10 MMPPLGC(me)AG 11 MMP ESPAYYTA 12 MMP RLQLKL 13 MMP RLQLKAC 14MMP2, MMP9, MMP14 EP(Cit)G(Hof)YL 15 Urokinase plasminogen SGRSA 16activator (uPA) Urokinase plasminogen DAFK 17 activator (uPA)Urokinase plasminogen GGGRR 18 activator (uPA) Lysosomal Enzyme GFLG 19Lysosomal Enzyme ALAL 20 Lysosomal Enzyme FK 21 Cathepsin B NLL 22Cathepsin D PIC(Et)FF 23 Cathepsin K GGPRGLPG 24Prostate Specific Antigen HSSKLQ 25 Prostate Specific Antigen HSSKLQL 26Prostate Specific Antigen HSSKLQEDA 27 Herpes Simplex Virus ProteaseLVLASSSFGY 28 HIV Protease GVSQNYPIVG 29 CMV Protease GVVQASCRLA 30Thrombin F(Pip)RS 31 Thrombin DPRSFL 32 Thrombin PPRSFL 33 Caspase-3DEVD 34 Caspase-3 DEVDP 35 Caspase-3 KGSGDVEG 36 Interleukin 1βconverting GWEHDG 37 enzyme Enterokinase EDDDDKA 38 FAP KQEQNPGST 39Kallikrein 2 GKAFRR 40 Plasmin DAFK 41 Plasmin DVLK 42 Plasmin DAFK 43TOP ALLLALL 44

Provided herein are pharmaceutical compositions comprising polypeptidesequences. As with all peptides, polypeptides, and proteins, includingfragments thereof, it is understood that additional modifications in theamino acid sequence of the chimeric polypeptides (amino acid sequencevariants) can occur that do not alter the nature or function of thepeptides, polypeptides, or proteins. Such modifications includeconservative amino acid substitutions and are discussed in greaterdetail below.

The compositions provided herein have a desired function. Thecompositions are comprised of at least an IL-2 polypeptide, a blockingmoiety, e.g., a steric blocking polypeptide, and an optional serumhalf-life extension element, and an optional targeting polypeptide, withone or more linkers connecting each polypeptide in the composition. Thefirst polypeptide, e.g., an IL-2 mutein, is provided to be an activeagent. The blocking moiety is provided to block the activity of theinterleukin. The linker polypeptide, e.g., a protease cleavablepolypeptide, is provided to be cleaved by a protease that isspecifically expressed at the intended target of the active agent.Optionally, the blocking moiety blocks the activity of the firstpolypeptide by binding the interleukin polypeptide. In some embodiments,the blocking moiety, e.g., a steric blocking peptide, is linked to theinterleukin via a protease-cleavable linker which is cleaved at the siteof action (e.g., by inflammation specific or tumor-specific proteases)releasing the cytokine full activity at the desired site.

The protease cleavage site may be a naturally occurring proteasecleavage site or an artificially engineered protease cleavage site. Theartificially engineered protease cleavage site can be cleaved by morethan one protease specific to the desired environment in which cleavagewill occur, e.g., a tumor. The protease cleavage site may be cleavableby at least one protease, at least two proteases, at least threeproteases, or at least four proteases.

In some embodiments, the linker comprises glycine-glycine, asortase-recognition motif, or a sortase-recognition motif and a peptidesequence (Gly₄Ser)_(n) (SEQ ID NO: 126) or (Gly₃Ser)_(n), (SEQ ID NO:127) wherein n is 1, 2, 3, 4 or 5. In one embodiment, thesortase-recognition motif comprises a peptide sequence LPXTG (SEQ ID NO:125), where X is any amino acid. In one embodiment, the covalent linkageis between a reactive lysine residue attached to the C-terminal of thecytokine polypeptide and a reactive aspartic acid attached to theN-terminal of the blocking or other moiety. In one embodiment, thecovalent linkage is between a reactive aspartic acid residue attached tothe N-terminal of the cytokine polypeptide and a reactive lysine residueattached to the C-terminal of the blocking or other moiety.

Cleavage and Inducibility

As described herein, the activity of the cytokine polypeptide thecontext of the fusion protein is attenuated, and protease cleavage atthe desired site of activity, such as in a tumor microenvironment,releases a form of the cytokine from the fusion protein that is muchmore active as a cytokine receptor agonist than the fusion protein. Forexample, the cytokine-receptor activating (agonist) activity of thefusion polypeptide can be at least about 10×, at least about 50×, atleast about 100×, at least about 250×, at least about 500×, or at leastabout 1000× less than the cytokine receptor activating activity of thecytokine polypeptide as a separate molecular entity. The cytokinepolypeptide that is part of the fusion protein exists as a separatemolecular entity when it contains an amino acid that is substantiallyidentical to the cytokine polypeptide and does not substantially includeadditional amino acids and is not associated (by covalent ornon-covalent bonds) with other molecules. If necessary, a cytokinepolypeptide as a separate molecular entity may include some additionalamino acid sequences, such as a tag or short sequence to aid inexpression and/or purification.

In other examples, the cytokine-receptor activating (agonist) activityof the fusion polypeptide is at least about 10×, at least about 50×, atleast about 100×, at least about 250×, at least about 500×, or about1000× less than the cytokine receptor activating activity of thepolypeptide that contains the cytokine polypeptide that is produced bycleavage of the protease-cleavable linker in the fusion protein. Inother words, the cytokine receptor activating (agonist) activity of thepolypeptide that contains the cytokine polypeptide that is produced bycleavage of the protease-cleavable linker in the fusion protein is atleast about 10×, at least about 50×, at least about 100×, at least about250×, at least about 500×, or at least about 1000× greater than thecytokine receptor activating activity of the fusion protein.

Polypeptide Substitutions

The polypeptides described herein can include components (e.g., thecytokine, the blocking moiety) that have the same amino acid sequence ofthe corresponding naturally occurring protein (e.g., IL-2, IL-15, HSA)or can have an amino acid sequence that differs from the naturallyoccurring protein so long as the desired function is maintained. It isunderstood that one way to define any known modifications andderivatives or those that might arise, of the disclosed proteins andnucleic acids that encode them is through defining the sequence variantsin terms of identity to specific known reference sequences. Specificallydisclosed are polypeptides and nucleic acids which have at least, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to thechimeric polypeptides provided herein. For example, provided arepolypeptides or nucleic acids that have at least, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99 percent identity to the sequence of any ofthe nucleic acids or polypeptides described herein. Those of skill inthe art readily understand how to determine the identity of twopolypeptides or two nucleic acids. For example, the identity can becalculated after aligning the two sequences so that the identity is atits highest level.

Another way of calculating identity can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the identity alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of identity can be obtained for nucleic acids by, forexample, the algorithms disclosed in Zuker, Science 244:48-52 (1989);Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989); Jaeger etal., Methods Enzymol. 183:281-306 (1989), which are herein incorporatedby reference for at least material related to nucleic acid alignment. Itis understood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

Protein modifications include amino acid sequence modifications.Modifications in amino acid sequence may arise naturally as allelicvariations (e.g., due to genetic polymorphism), may arise due toenvironmental influence (e.g., by exposure to ultraviolet light), or maybe produced by human intervention (e.g., by mutagenesis of cloned DNAsequences), such as induced point, deletion, insertion and substitutionmutants. These modifications can result in changes in the amino acidsequence, provide silent mutations, modify a restriction site, orprovide other specific mutations. Amino acid sequence modificationstypically fall into one or more of three classes: substitutional,insertional or deletional modifications. Insertions include amino and/orcarboxyl terminal fusions as well as intrasequence insertions of singleor multiple amino acid residues. Insertions ordinarily will be smallerinsertions than those of amino or carboxyl terminal fusions, forexample, on the order of one to four residues. Deletions arecharacterized by the removal of one or more amino acid residues from theprotein sequence. Typically, no more than about from 2 to 6 residues aredeleted at any one site within the protein molecule Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional modifications are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTable 2 and are referred to as conservative substitutions.

TABLE 2 Exemplary amino acid substitutions Amino Acid ExemplarySubstitutions Ala Ser, Gly, Cys Arg Lys, Gln, Met, Ile Asn Gln, His,Glu, Asp Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln Asn, Lys, Glu, Asp GluAsp, Asn, Gln Gly Pro, Ala His Asn, Gln Ile Leu, Val, Met Leu Ile, Val,Met Lys Arg, Gln, Met, Ile Met Leu, Ile, Val Phe Met, Leu, Tyr, Trp, HisSer Thr, Met, Cys Thr Ser, Met, Val Trp Tyr, Phe Tyr Trp, Phe, His ValIle, Leu, Met

Modifications, including the specific amino acid substitutions, are madeby known methods. For example, modifications are made by site specificmutagenesis of nucleotides in the DNA encoding the polypeptide, therebyproducing DNA encoding the modification, and thereafter expressing theDNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis.

Modifications can be selected to optimize binding. For example, affinitymaturation techniques can be used to alter binding of the scFv byintroducing random mutations inside the complementarity determiningregions (CDRs). Such random mutations can be introduced using a varietyof techniques, including radiation, chemical mutagens or error-pronePCR. Multiple rounds of mutation and selection can be performed using,for example, phage display.

The disclosure also relates to nucleic acids that encode the chimericpolypeptides described herein, and to the use of such nucleic acids toproduce the chimeric polypeptides and for therapeutic purposes. Forexample, the invention includes DNA and RNA molecules (e.g., mRNA,self-replicating RNA) that encode a chimeric polypeptide and to thetherapeutic use of such DNA and RNA molecules.

Exemplary Compositions

Exemplary fusion proteins of the invention combine the above describedelements in a variety of orientations. The orientations described inthis section are meant as examples and are not to be consideredlimiting.

In some embodiments, the fusion protein comprises an IL-2 polypeptide, ablocking moiety and a half-life extension element. In some embodiments,the IL-2 polypeptide is positioned between the half-life extensionelement and the blocking moiety. In some embodiments, the IL-2polypeptide is N-terminal to the blocking moiety and the half-lifeextension element. In some such embodiments, the IL-2 polypeptide isproximal to the blocking moiety; in some such embodiments, the IL-2polypeptide is proximal to the half-life extension element. At least oneprotease-cleavable linker must be included in all embodiments, such thatthe IL-2 polypeptide may be active upon cleavage. In some embodiments,the IL-2 polypeptide is C-terminal to the blocking moiety and thehalf-life extension element. Additional elements may be attached to oneanother by a cleavable linker, a non-cleavable linker, or by directfusion. In some cases it is beneficial to include two of the samecytokine to facilitate dimerization.

In some embodiments, the blocking domains used are capable of extendinghalf-life, and the IL-2 polypeptide is positioned between two suchblocking domains. In some embodiments, the IL-2 polypeptide ispositioned between two blocking domains, one of which is capable ofextending half-life.

In some embodiments, two cytokines are included in the same construct,at least one being IL-2. In some embodiments, the cytokines areconnected to two blocking domains each (three in total in one molecule),with a blocking domain between the two cytokine domains. In someembodiments, one or more additional half-life extension domains may beincluded to optimize pharmacokinetic properties.

In some embodiments, three cytokines are included in the same construct.In some embodiments, the third cytokine may function to block the othertwo in place of a blocking domain between the two cytokines.

Preferred half-life extension elements for use in the fusion proteinsare human serum albumin (HSA), an antibody or antibody fragment (e.g.,scFV, dAb) which binds serum albumin, a human or humanized IgG, or afragment of any of the foregoing. In some preferred embodiments, theblocking moiety is human serum albumin (HSA), or an antibody or antibodyfragment which binds serum albumin, an antibody which binds the cytokineand prevents activation of binding or activation of the cytokinereceptor, another cytokine, or a fragment of any of the foregoing. Inpreferred embodiments comprising an additional targeting domain, thetargeting domain is an antibody which binds a cell surface protein whichis enriched on the surface of cancer cells, such as EpCAM, FOLR1, andFibronectin.

Methods of Treatment and Pharmaceutical Compositions

Further provided are methods of treating a subject with or at risk ofdeveloping an of a disease or disorder, such as proliferative disease, atumorous disease, an inflammatory disease, an immunological disorder, anautoimmune disease, an infectious disease, a viral disease, an allergicreaction, a parasitic reaction, or graft-versus-host disease. Themethods administering to a subject in need thereof an effective amountof a fusion protein as disclosed herein that is typically administeredas a pharmaceutical composition. In some embodiments, the method furthercomprises selecting a subject with or at risk of developing such adisease or disorder. The pharmaceutical composition preferably comprisesa blocked cytokine, fragment or mutein thereof that is activated at asite of inflammation or a tumor. In one embodiment, the chimericpolypeptide comprises a cytokine polypeptide, fragment or mutein thereofand a serum half-life extension element. In another embodiment, thechimeric polypeptide comprises a cytokine polypeptide, fragment ormutein thereof and a blocking moiety, e.g., a steric blockingpolypeptide, wherein the steric blocking polypeptide is capable ofsterically blocking the activity of the cytokine polypeptide, fragmentor mutein thereof. In another embodiment, the chimeric polypeptidecomprises a cytokine polypeptide, fragment or mutein thereof, a blockingmoiety, and a serum half-life extension element.

Inflammation is part of the complex biological response of body tissuesto harmful stimuli, such as pathogens, damaged cells, or irritants, andis a protective response involving immune cells, blood vessels, andmolecular mediators. The function of inflammation is to eliminate theinitial cause of cell injury, clear out necrotic cells and tissuesdamaged from the original insult and the inflammatory process, and toinitiate tissue repair. Inflammation can occur from infection, as asymptom or a disease, e.g., cancer, atherosclerosis, allergies,myopathies, HIV, obesity, or an autoimmune disease. An autoimmunedisease is a chronic condition arising from an abnormal immune responseto a self-antigen. Autoimmune diseases that may be treated with thepolypeptides disclosed herein include but are not limited to lupus,celiac disease, diabetes mellitus type 1, Graves disease, inflammatorybowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, andsystemic lupus erythematosus.

The pharmaceutical composition can comprise one or moreprotease-cleavable linker sequences. The linker sequence serves toprovide flexibility between polypeptides, such that each polypeptide iscapable of inhibiting the activity of the first polypeptide. The linkersequence can be located between any or all of the cytokine polypeptide,fragment or mutein thereof, the blocking moiety, and serum half-lifeextension element. Optionally, the composition comprises, two, three,four, or five linker sequences. The linker sequence, two, three, or fourlinker sequences can be the same or different linker sequences. In oneembodiment, the linker sequence comprises GGGGS (SEQ ID NO: 132), GSGSGS(SEQ ID NO: 133), or G(SGGG)₂SGGT (SEQ ID NO: 134). In anotherembodiment, the linker comprises a protease-cleavable sequence selectedfrom group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO:128), IPVSLRSG (SEQ ID NO: 129), VPLSLYSG (SEQ ID NO: 130), andSGESPAYYTA (SEQ ID NO: 131).

In some embodiments, the linker is cleaved by a protease selected fromthe group consisting of a kallikrein, thrombin, chymase,carboxypeptidase A, cathepsin G, an elastase, PR-3, granzyme M, acalpain, a matrix metalloproteinase (MMP), a plasminogen activator, acathepsin, a caspase, a tryptase, or a tumor cell surface protease.

Suitable linkers can be of different lengths, such as from 1 amino acid(e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids,from 3 amino acids to 12 amino acids, including 4 amino acids to 10amino acids, amino acids to 9 amino acids, 6 amino acids to 8 aminoacids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60amino acids.

Further provided are methods of treating a subject with or at risk ofdeveloping cancer. The methods comprise administering to the subject inneed thereof an effective amount of a chimeric polypeptide (a fusionprotein) as disclosed herein that is typically administered as apharmaceutical composition. In some embodiments, the method furthercomprises selecting a subject with or at risk of developing cancer. Thepharmaceutical composition preferably comprises a blocked cytokine,fragment or mutein thereof that is activated at a tumor site.Preferably, the tumor is a solid tumor. The cancer may be, but is notlimited to, a colon cancer, a lung cancer, a melanoma, a sarcoma, arenal cell carcinoma, and a breast cancer.

The method can further involve the administration of one or moreadditional agents to treat cancer, such as chemotherapeutic agents(e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, Oncovin,Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine,Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-L1,anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cellular therapies (e.g.,CAR-T, T-cell therapy), oncolytic viruses and the like.

Provided herein are pharmaceutical formulations or compositionscontaining the chimeric polypeptides and a pharmaceutically acceptablecarrier. The herein provided compositions are suitable foradministration in vitro or in vivo. By pharmaceutically acceptablecarrier is meant a material that is not biologically or otherwiseundesirable, i.e., the material is administered to a subject withoutcausing undesirable biological effects or interacting in a deleteriousmanner with the other components of the pharmaceutical formulation orcomposition in which it is contained. The carrier is selected tominimize degradation of the active ingredient and to minimize adverseside effects in the subject.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, David B. Troy, ed.,Lippicott Williams & Wilkins (2005). Typically, an appropriate amount ofa pharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic, although the formulate can be hypertonic orhypotonic if desired. Examples of the pharmaceutically-acceptablecarriers include, but are not limited to, sterile water, saline,buffered solutions like Ringer's solution, and dextrose solution. The pHof the solution is generally about 5 to about 8 or from about 7 to 7.5.Other carriers include sustained release preparations such assemipermeable matrices of solid hydrophobic polymers containing theimmunogenic polypeptides. Matrices are in the form of shaped articles,e.g., films, liposomes, or microparticles. Certain carriers may be morepreferable depending upon, for instance, the route of administration andconcentration of composition being administered. Carriers are thosesuitable for administration of the chimeric polypeptides or nucleic acidsequences encoding the chimeric polypeptides to humans or othersubjects.

The pharmaceutical formulations or compositions are administered in anumber of ways depending on whether local or systemic treatment isdesired and on the area to be treated. The compositions are administeredvia any of several routes of administration, including topically,orally, parenterally, intravenously, intra-articularly,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, intrahepatically, intracranially,nebulization/inhalation, or by installation via bronchoscopy. In someembodiments, the compositions are administered locally(non-systemically), including intratumorally, intra-articularly,intrathecally, etc.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives are optionally present suchas, for example, antimicrobials, anti-oxidants, chelating agents, andinert gases and the like.

Formulations for topical administration include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, and powders.Conventional pharmaceutical carriers, aqueous, powder, or oily bases,thickeners and the like are optionally necessary or desirable.

Compositions for oral administration include powders or granules,suspension or solutions in water or non-aqueous media, capsules,sachets, or tables. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders are optionally desirable.

Optionally, the chimeric polypeptides or nucleic acid sequences encodingthe chimeric polypeptides are administered by a vector. There are anumber of compositions and methods which can be used to deliver thenucleic acid molecules and/or polypeptides to cells, either in vitro orin vivo via, for example, expression vectors. These methods andcompositions can largely be broken down into two classes: viral baseddelivery systems and non-viral based delivery systems. Such methods arewell known in the art and readily adaptable for use with thecompositions and methods described herein. Such compositions and methodscan be used to transfect or transduce cells in vitro or in vivo, forexample, to produce cell lines that express and preferably secrete theencoded chimeric polypeptide or to therapeutically deliver nucleic acidsto a subject. The components of the chimeric nucleic acids disclosedherein typically are operably linked in frame to encode a fusionprotein.

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids into the cell without degradation and include apromoter yielding expression of the nucleic acid molecule and/orpolypeptide in the cells into which it is delivered. Viral vectors are,for example, Adenovirus, Adeno-associated virus, herpes virus, Vacciniavirus, Polio virus, Sindbis, and other RNA viruses, including theseviruses with the HIV backbone. Also preferred are any viral familieswhich share the properties of these viruses which make them suitable foruse as vectors. Retroviral vectors, in general are described by Coffinet al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), whichis incorporated by reference herein for the vectors and methods ofmaking them. The construction of replication-defective adenoviruses hasbeen described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie etal., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol.57:267-74 (1986); Davidson et al., J. Virol. 61:1226-39 (1987); Zhang etal., BioTechniques 15:868-72 (1993)). The benefit and the use of theseviruses as vectors is that they are limited in the extent to which theycan spread to other cell types, since they can replicate within aninitial infected cell, but are unable to form new infectious viralparticles. Recombinant adenoviruses have been shown to achieve highefficiency after direct, in vivo delivery to airway epithelium,hepatocytes, vascular endothelium, CNS parenchyma, and a number of othertissue sites. Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

The provided polypeptides and/or nucleic acid molecules can be deliveredvia virus like particles. Virus like particles (VLPs) consist of viralprotein(s) derived from the structural proteins of a virus. Methods formaking and using virus like particles are described in, for example,Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

The provided polypeptides can be delivered by subviral dense bodies(DBs). DBs transport proteins into target cells by membrane fusion.Methods for making and using DBs are described in, for example,Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

The provided polypeptides can be delivered by tegument aggregates.Methods for making and using tegument aggregates are described inInternational Publication No. WO 2006/110728.

Non-viral based delivery methods, can include expression vectorscomprising nucleic acid molecules and nucleic acid sequences encodingpolypeptides, wherein the nucleic acids are operably linked to anexpression control sequence. Suitable vector backbones include, forexample, those routinely used in the art such as plasmids, artificialchromosomes, BACs, YACs, or PACs. Numerous vectors and expressionsystems are commercially available from such corporations as Novagen(Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla,Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.). Vectorstypically contain one or more regulatory regions. Regulatory regionsinclude, without limitation, promoter sequences, enhancer sequences,response elements, protein recognition sites, inducible elements,protein binding sequences, 5′ and 3′ untranslated regions (UTRs),transcriptional start sites, termination sequences, polyadenylationsequences, and introns. Such vectors can also be used to make thechimeric polypeptides by expression is a suitable host cell, such as CHOcells.

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis B virus, and most preferably cytomegalovirus(CMV), or from heterologous mammalian promoters, e.g., β-actin promoteror EF1α promoter, or from hybrid or chimeric promoters (e.g., CMVpromoter fused to the β-actin promoter). Of course, promoters from thehost cell or related species are also useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′ or3′ to the transcription unit. Furthermore, enhancers can be within anintron as well as within the coding sequence itself. They are usuallybetween 10 and 300 base pairs (bp) in length, and they function in cis.Enhancers usually function to increase transcription from nearbypromoters. Enhancers can also contain response elements that mediate theregulation of transcription. While many enhancer sequences are knownfrom mammalian genes (globin, elastase, albumin, fetoprotein, andinsulin), typically one will use an enhancer from a eukaryotic cellvirus for general expression. Preferred examples are the SV40 enhanceron the late side of the replication origin, the cytomegalovirus earlypromoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

The promoter and/or the enhancer can be inducible (e.g., chemically orphysically regulated). A chemically regulated promoter and/or enhancercan, for example, be regulated by the presence of alcohol, tetracycline,a steroid, or a metal. A physically regulated promoter and/or enhancercan, for example, be regulated by environmental factors, such astemperature and light. Optionally, the promoter and/or enhancer regioncan act as a constitutive promoter and/or enhancer to maximize theexpression of the region of the transcription unit to be transcribed. Incertain vectors, the promoter and/or enhancer region can be active in acell type specific manner. Optionally, in certain vectors, the promoterand/or enhancer region can be active in all eukaryotic cells,independent of cell type. Preferred promoters of this type are the CMVpromoter, the SV40 promoter, the β-actin promoter, the EF1α promoter,and the retroviral long terminal repeat (LTR).

The vectors also can include, for example, origins of replication and/ormarkers. A marker gene can confer a selectable phenotype, e.g.,antibiotic resistance, on a cell. The marker product is used todetermine if the vector has been delivered to the cell and oncedelivered is being expressed. Examples of selectable markers formammalian cells are dihydrofolate reductase (DHFR), thymidine kinase,neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin.When such selectable markers are successfully transferred into amammalian host cell, the transformed mammalian host cell can survive ifplaced under selective pressure. Examples of other markers include, forexample, the E. coli lacZ gene, green fluorescent protein (GFP), andluciferase. In addition, an expression vector can include a tag sequencedesigned to facilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such as GFP,glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, orFLAG™ tag (Kodak; New Haven, Conn.) sequences typically are expressed asa fusion with the encoded polypeptide. Such tags can be insertedanywhere within the polypeptide including at either the carboxyl oramino terminus.

As used herein, the terms peptide, polypeptide, or protein are usedbroadly to mean two or more amino acids linked by a peptide bond.Protein, peptide, and polypeptide are also used herein interchangeablyto refer to amino acid sequences. It should be recognized that the termpolypeptide is not used herein to suggest a particular size or number ofamino acids comprising the molecule and that a peptide of the inventioncan contain up to several amino acid residues or more. As usedthroughout, subject can be a vertebrate, more specifically a mammal(e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit,rat, and guinea pig), birds, reptiles, amphibians, fish, and any otheranimal. The term does not denote a particular age or sex. Thus, adultand newborn subjects, whether male or female, are intended to becovered. As used herein, patient or subject may be used interchangeablyand can refer to a subject with a disease or disorder (e.g., cancer).The term patient or subject includes human and veterinary subjects.

A subject at risk of developing a disease or disorder can be geneticallypredisposed to the disease or disorder, e.g., have a family history orhave a mutation in a gene that causes the disease or disorder, or showearly signs or symptoms of the disease or disorder. A subject currentlywith a disease or disorder has one or more than one symptom of thedisease or disorder and may have been diagnosed with the disease ordisorder.

The methods and agents as described herein are useful for bothprophylactic and therapeutic treatment. For prophylactic use, atherapeutically effective amount of the chimeric polypeptides orchimeric nucleic acid sequences encoding the chimeric polypeptidesdescribed herein are administered to a subject prior to onset (e.g.,before obvious signs of cancer or inflammation) or during early onset(e.g., upon initial signs and symptoms of cancer or inflammation).Prophylactic administration can occur for several days to years prior tothe manifestation of symptoms of cancer or inflammation. Prophylacticadministration can be used, for example, in the preventative treatmentof subjects diagnosed with a genetic predisposition to cancer.Therapeutic treatment involves administering to a subject atherapeutically effective amount of the chimeric polypeptides or nucleicacid sequences encoding the chimeric polypeptides described herein afterdiagnosis or development of cancer or inflammation (e.g., an autoimmunedisease). Prophylactic use may also apply when a patient is undergoing atreatment, e.g., a chemotherapy, in which inflammation is expected.

According to the methods taught herein, the subject is administered aneffective amount of the agent (e.g., a chimeric polypeptide). The termseffective amount and effective dosage are used interchangeably. The termeffective amount is defined as any amount necessary to produce a desiredphysiologic response. Effective amounts and schedules for administeringthe agent may be determined empirically, and making such determinationsis within the skill in the art. The dosage ranges for administration arethose large enough to produce the desired effect in which one or moresymptoms of the disease or disorder are affected (e.g., reduced ordelayed). The dosage should not be so large as to cause substantialadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex, type of disease, the extent of the disease or disorder,route of administration, or whether other drugs are included in theregimen, and can be determined by one of skill in the art. The dosagecan be adjusted by the individual physician in the event of anycontraindications. Dosages can vary and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products.

As used herein the terms treatment, treat, or treating refers to amethod of reducing the effects of a disease or condition or symptom ofthe disease or condition. Thus, in the disclosed method, treatment canrefer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%reduction in the severity of an established disease or condition orsymptom of the disease or condition. For example, a method for treatinga disease is considered to be a treatment if there is a 10% reduction inone or more symptoms of the disease in a subject as compared to acontrol. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or any percent reduction in between 10% and 100% ascompared to native or control levels. It is understood that treatmentdoes not necessarily refer to a cure or complete ablation of thedisease, condition, or symptoms of the disease or condition.

As used herein, the terms prevent, preventing, and prevention of adisease or disorder refers to an action, for example, administration ofthe chimeric polypeptide or nucleic acid sequence encoding the chimericpolypeptide, that occurs before or at about the same time a subjectbegins to show one or more symptoms of the disease or disorder, whichinhibits or delays onset or exacerbation of one or more symptoms of thedisease or disorder. As used herein, references to decreasing, reducing,or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or greater as compared to a control level. Such terms caninclude but do not necessarily include complete elimination.

IL-2 variants have been developed that are selective for IL-2Rαβγrelative to IL-2Rβγ (Shanafelt, A. B., et al., 2000, NatBiotechnol.18:1197-202; Cassell, D. J., et. al., 2002, Curr Pharm Des.,8:2171-83). These variants have amino acid substitutions, which reducetheir affinity for IL-2Rβ. Because IL-2 has undetectable affinity forIL-2Rγ, these variants consequently have reduced affinity for theIL-2Rβγ receptor complex and reduced ability to activateIL-2Rβγ-expressing cells, but retain the ability to bind IL-2Rα and theability to bind and activate the IL-2Rαβγ receptor complex.

One of these variants, IL-2/N88R (Bay 50-4798), was clinically tested asa low-toxicity version of IL-2 as an immune system stimulator, based onthe hypothesis that IL-2Rβγ-expressing NK cells are a major contributorto toxicity. Bay 50-4798 was shown to selectively stimulate theproliferation of activated T cells relative to NK cells, and wasevaluated in phase I/II clinical trials in cancer patients (Margolin,K., et. al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey,R. T., et. al., 2008, J Interferon Cytokine Res., 28:89-100). Theseclinical trials showed that Bay 50-4798 was considerably safer and moretolerable than aldesleukin, and also showed that it increased the levelsof CD4+CD25+ T cells, a cell population enriched in Treg cells.Subsequent to these trials, research in the field more fully establishedthe identity of Treg cells and demonstrated that Treg cells selectivelyexpress IL-2Rαβγ (reviewed in Malek, T. R., et al., 2010, Immunity,33:153-65).

In addition, mutants can be made that selectively alter the affinity forthe CD25 chain relative to native IL-2.

IL-2 can be engineered to produce mutants that bind the IL-2R complexgenerally or the IL-2Rα subunit specifically with an affinity thatdiffers from that of the corresponding wild-type IL-2 or of a presentlyavailable mutant (referred to as C125S, as the cysteine residue atposition 125 is replaced with a serine residue).

Accordingly, the present invention features mutant interleukin-2 (IL-2*)polypeptides that include an amino acid sequence that is at least 80%identical to wild-type IL-2 (e.g., 85, 87, 90, 95, 97, 98, or 99%identical) and that bind, as compared to WT IL-2, with higher to theIL-2 trimeric receptor relative to the dimeric IL-2 receptor. Typically,the muteins will also bind an IL-2 receptor a subunit (IL-2Ra) with anaffinity that is greater than the affinity with which wild type IL-2binds the IL-2Ra. The amino acid sequence within mutant IL-2polypeptides can vary from SEQ ID NO: 1 (UniProtKB accession numberP60568) by virtue of containing (or only containing) one or more aminoacid substitutions, which may be considered conservative ornon-conservative substitutions. Non-naturally occurring amino acids canalso be incorporated. Alternatively, or in addition, the amino acidsequence can vary from SEQ ID NO: 1 (which may be considered the“reference” sequence) by virtue of containing and addition and/ordeletion of one or more amino acid residues. More specifically, theamino acid sequence can differ from that of SEQ ID NO:1 by virtue of amutation at least one of the following positions of SEQ ID NO:1: 1, 4,8, 9, 10, 11, 13, 15, 26, 29, 30, 31, 35, 37, 46, 48, 49, 54, 61, 64,67, 68, 69, 71, 73, 74, 75, 76, 79, 88, 89, 90, 92, 99, 101, 103, 114,125, 128, or 133 (or combinations thereof). As noted, as few as one ofthese positions may be altered, as may two, three, four, five, six,seven, eight, nine, ten, or 11 or more (including up to all) of thepositions. For example, the amino acid sequence can differ from SEQ IDNO: 1 at positions 69 and 74 and further at one or more of positions 30,35, and 128. The amino acid sequence can also differ from SEQ ID NO:2(as disclosed in U.S. Pat. No. 7,569,215, incorporated herein byreference) at one of the following sets of positions: (a) positions 64,69, and 74; (b) positions 69, 74, and 101; (c) positions 69, 74, and128; (d) positions 30, 69, 74, and 103; (e) positions 49, 69, 73, and76; (f) positions 69, 74, 101, and 133; (g) positions 30, 69, 74, and128; (h) positions 69, 74, 88, and 99; (i) positions 30, 69, 74, and128; (j) positions 9, 11, 35, 69, and 74; (k) positions 1, 46, 49, 61,69, and 79; (l) positions 48, 68, 71, 90, 103, and 114; (m) positions 4,10, 11, 69, 74, 88, and 133; (n) positions 15, 30 31, 35, 48, 69, 74,and 92; (o) positions 30, 68, 69, 71, 74, 75, 76, and 90; (p) positions30, 31, 37, 69, 73, 74, 79, and 128; (q) positions 26, 29, 30, 54, 67,69, 74, and 92; (r) positions 8, 13, 26, 30, 35, 37, 69, 74, and 92; and(s) positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 89. Aside frommutations at these positions, the amino acid sequence of the mutant IL-2polypeptide can otherwise be identical to SEQ ID NO: 1. With respect tospecific substitutions, the amino acid sequence can differ from SEQ IDNO: 1 by virtue of having one or more of the following mutations: A1T,S4P, K8R, K9T, T10A, Q11R, Q13R, E15K, N26D, N29S, N30S, N30D, N30T,Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R, K49E, K54R, E61D, K64R,E67G, E68D, V69A, N71T, N71A, N71R, A73V, Q74P, 575P, K76E, K76R, H79R,N88D, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, I128A, T133A,or T133N. Our nomenclature is consistent with that of the scientificliterature, where the single letter code of the amino acid in thewild-type or reference sequence is followed by its position within thesequence and then by the single letter code of the amino acid with whichit is replaced. Thus, A1T designates a substitution of the alanineresidue a position 1 with threonine. Other mutant polypeptides withinthe scope of the invention include those that include a mutant of SEQ IDNO: 2 having substitutions at V69 (e.g., A) and Q74 (e.g., P). Forexample, the amino acid sequence can include one of the following setsof mutations with respect to SEQ ID NO:2: (a) K64R, V69A, and Q74P; (b)V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P,and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, andT133N; (g) N305, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, and S99P;(i) N305, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V69A, and Q74P;(k) A1T, M46L, K49R, E61D, V69A, and H79R; (1) K48E, E68D, N71T, N90H,F103S, and I114V; (m) S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A; (n)E15K, N305 Y31H, K35R, K48E, V69A, Q74P, and I92T; (o) N305, E68D, V69A,N71A, Q74P, 575P, K76R, and N90H; (p) N30S, Y31C, T37A, V69A, A73V,Q74P, H79R, and I128T; (q) N26D, N29S, N30S, K54R, E67G, V69A, Q74P, andI92T; (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T; and(s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V. SEQID NO: 2 is disclosed in U.S. Pat. No. 7,569,215, which is incorporatedherein by reference as an exemplary IL-2 polypeptide sequence that canbe used in the invention.

As noted above, any of the mutant IL-2 polypeptides disclosed herein caninclude the sequences described; they can also be limited to thesequences described and otherwise identical to SEQ ID NO: 1. Moreover,any of the mutant IL-2 polypeptides described herein can optionallyinclude a substitution of the cysteine residue at position 125 withanother residue (e.g., serine) and/or can optionally include a deletionof the alanine residue at position 1 of SEQ ID NO: 1.

The mutant IL-2 polypeptides disclosed herein can bind to the IL-2Rαsubunit with a K_(d) of less than about 28 nM (e.g., less than about 25nM; less than about 5 nM; about 1 nM; less than about 500 pM; or lessthan about 100 pM). More specifically, a mutant IL-2 polypeptide canhave an affinity equilibrium constant less than 1.0 nM (e.g., about 0.8,0.6, 0.4, or 0.2 nM). Affinity can also be expressed as a relative rateof dissociation from an IL-2Rα subunit or from an IL-2 receptor complex(e.g., a complex expressed on the surface of a cell or otherwisemembrane bound). For example, the mutant IL-2 polypeptides candissociate from, e.g., IL-2Ra, at a decreased rate relative to awild-type polypeptide or to an IL-2 based therapeutic, e.g., IL-2*.Alternatively, affinity can be characterized as the time, or averagetime, an IL-2* polypeptide persists on, for example, the surface of acell expressing an IL-2R. For example, an IL-2*polypeptide can persiston the receptor for at least about 2, 5, 10, 50, 100, or 250 times (ormore).

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided herein.

Example 1: Detection of IL-2, IL-2 Mutein, IL-2Rα and IL-2Rγ in FusionProteins by ELISA

IL-2 or IL-2 mutein in a fusion protein is detected with a commerciallyavailable antibody, e.g., the anti-IL-2 monoclonal (JES6-1A12) (BDPharmingen; San Jose, Calif.). A positive control is used to showwhether the monoclonal antibody recognizes the cytokine or mutein.Antibodies against IL-2Rα and IL-2Rγ chain are also used. Wells of a96-well plate are coated with an antibody (2.5 μg/ml) in PBS. Wells areblocked with 5% non-fat milk in PBS with 0.2% Tween®20 (PBS-M-Tw) andfusion proteins are added for 1-2 hours at 37° C. After washing, ananti-IL-2 biotin-labeled antibody, e.g., JES5H4 (BD Pharmingen) is addedand binding is detected using Strepavidin HRP (Southern BiotechnologyAssociates; Birmingham, Ala.). The ELISA plate is developed by adding 50μl O-phenylenediamine (OPD) (Sigma-Aldrich) in 0.1M Citrate pH 4.5 and0.04% H₂O₂, stopped by adding 50 μl/well 2N H₂SO₄ and the absorbance wasread at 490 nm.

Example 2: Protease Cleavage of IL-2 Fusion Protein by MMP9 Protease

One of skill in the art would be familiar with methods of setting upprotein cleavage assay. 100 ug of protein in 1×PBS pH 7.4 were cleavedwith 1 ug active MMP9 (Sigma catalog # SAE0078-50 or Enzo catalogBML-SE360) and incubated at room temperature for up to 16 hours.Digested protein is subsequently used in functional assays or stored at−80° C. prior to testing. Extent of cleavage was monitored by SDS PAGEusing methods well known in the art. As shown in FIG. 10, the ACP16fusion protein was cleaved by MMP9 protease.

Example 3: CTLL-2 Assay

CTLL2 cells (ATCC) were plated in suspension at a concentration of500,000 cells/well in culture media with or without 40 mg/ml human serumalbumin (HSA) and stimulated with a dilution series of recombinant hIL-2or activatable hIL-2 for 72 hours at 37° C. and 5% CO₂. Activity ofuncleaved and cleaved activatable hIL-2 was tested. Cleaved activatablehIL-2 was generated by incubation with active MMP9. Cell activity wasassessed using a CellTiter-Glo (Promega) luminescence-based cellviability assay. Results are shown in FIGS. 7a-7h, 8a-8f, and 9a -9 z.

Example 4: Protease Cleavage of the IL-2/IL-2Ra/IL-2Rγ ChimericPolypeptide Results in Increased Accessibility to Antibodies andBiologically Active IL-2 Mutein

The IL-2 mutein fusion proteins are biochemically characterized beforeand after cleavage with a protease, e.g., PSA. Immunoblot analyses willshow that the fusion proteins can be cleaved by PSA and that there is anincrease in intensity of the predicted low molecular weight cleavageproduct of approximately 20 kDa reactive with an anti-IL-2 antibodyafter treatment of the samples with PSA. The degree of cleavage isdependent upon the amount of PSA as well as the time of incubation.Interestingly, when the fusion protein is analyzed before and after PSAtreatment by ELISA, it was found that the apparent amount of IL-2 isincreased after PSA cleavage. In this experiment, there is anapproximately 2 or 4-fold increase in the apparent amount of IL-2detected using this sandwich ELISA depending on the construct,suggesting that the antibody binding is partially hindered in the intactfusion protein. Aliquots of the same samples are also analyzed after PSAtreatment using the CTLL-2 cell line that requires IL-2 for growth andsurvival and the viability of cells can be ascertained using thecolorimetric MTT assay. In this assay, the more a supernatant can bediluted, the more biologically active IL-2 it contains, and there is anincrease in the amount of biologically active IL-2 after PSA cleavage.The amount of IL-2 mutein increase will suggest that after PSA cleavagethere is an increase in the predicted low molecular weight cleavagefragment of approximately 20 kDa reactive with an anti-IL-2 antibody, anincrease in antibody accessibility, and most importantly, an increase inthe amount of biologically active IL-2 mutein.

Example 5. In Vivo Delivery of a Protease Activated IL-2 Fusion ProteinResults in Decreased Tumor Growth

The chimeric polypeptide is examined to determine if it could havebiological effects in vivo. For these experiments a system is used inwhich tumor cells injected intraperitoneally rapidly and preferentiallyattach and grow initially on the milky spots, a series of organizedimmune aggregates found on the omentum (Gerber et al., Am. J. Pathol.169:1739-52 (2006)). This system offers a convenient way to examine theeffects of fusion protein treatment on tumor growth since fusionproteins can be delivered intraperitoneally multiple times and tumorgrowth can be analyzed by examining the dissociated omental cells. Forthese experiments, the Colon 38 cell line, a rapidly growing tumor cellline that expresses both MMP2 and MMP9 in vitro, may be used. Theomental tissue normally expresses a relatively small amount of MMP2 andMMP9, but, when Colon 38 tumor is present on the omentum, MMP levelsincrease. Using this tumor model, the ability of IL-2 mutein fusionproteins to affect tumor growth is examined. Colon 38 cells are injectedintraperitoneally, allowed to attach and grow for 1 day, and thentreated daily with fusion protein interaperitoneally. At day 7, theanimals are sacrificed and the omenta examined for tumor growth usingflow cytometry and by a colony-forming assay.

Example 6: Construction of an Exemplary Activatable IL-2 ProteinTargeting CD20 Generation of an Activatable IL-2 Domain

An IL-2 polypeptide capable of binding to CD20 polypeptide present in atumor or on a tumor cell is produced as follows. A nucleic acid isproduced that contains nucleic acid sequences: (1) encoding an IL-2polypeptide sequence and (2) one or more polypeptide linkers.Activatable IL-2 plasmid constructs can have optional Flag, His or otheraffinity tags, and are electroporated into HEK293 or other suitablehuman or mammalian cell lines and purified. Validation assays include Tcell activation assays using T cells responsive to IL-2 stimulation inthe presence of a protease.

Generation of a scFv CD20 Binding Domain

CD20 is one of the cell surface proteins present on B-lymphocytes. CD20antigen is found in normal and malignant pre-B and mature B lymphocytes,including those in over 90% of B-cell non-Hodgkin's lymphomas (NHL). Theantigen is absent in hematopoietic stem cells, activated B lymphocytes(plasma cells) and normal tissue. As such, several antibodies mostly ofmurine origin have been described: 1F5, 2B8/C2B8, 2H7, and 1H4.

Human or humanized anti-CD20 antibodies are therefore used to generatescFv sequences for CD20 binding domains of an activatable IL-2 protein.DNA sequences coding for human or humanized VL and VH domains areobtained, and the codons for the constructs are, optionally, optimizedfor expression in cells from Homo sapiens. The order in which the VL andVH domains appear in the scFv is varied (i.e., VL-VH, or VH-VLorientation), and three copies of the “G4S” or “G4S” subunit (G4S)₃connect the variable domains to create the scFv domain. Anti-CD20 scFvplasmid constructs can have optional Flag, His or other affinity tags,and are electroporated into HEK293 or other suitable human or mammaliancell lines and purified. Validation assays include binding analysis byFACS, kinetic analysis using Proteon, and staining of CD20-expressingcells.

Cloning of DNA Expression Constructs Encoding the Activatable IL-2Protein

The activatable IL-2 construct with protease cleavage site domains areused to construct an activatable IL-2 protein in combination with ananti-CD20 scFv domain and a serum half-life extension element (e.g., aHSA binding peptide or VH domain). For expression of an activatable IL-2protein in CHO cells, coding sequences of all protein domains are clonedinto a mammalian expression vector system. In brief, gene sequencesencoding the activatable IL-2 domain, serum half-life extension element,and CD20 binding domain along with peptide linkers L1 and L2 areseparately synthesized and subcloned. The resulting constructs are thenligated together in the order of CD20 binding domain-L1-IL-2 subunit1-L2-protease cleavage domain-L3-IL-2 subunit 2-L4-anti-CD20scFv-L5-serum half-life extension element to yield a final construct.All expression constructs are designed to contain coding sequences foran N-terminal signal peptide and a C-terminal hexahistidine (6×His)-tagto facilitate protein secretion and purification, respectively.

Expression of Activatable IL-2 Proteins in Stably Transfected CHO Cells

A CHO cell expression system (Flp-In®, Life Technologies), a derivativeof CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck,Proc. Natl. Acad Sci USA 1968; 60(4):1275-81), is used. Adherent cellsare subcultured according to standard cell culture protocols provided byLife Technologies.

For adaption to growth in suspension, cells are detached from tissueculture flasks and placed in serum-free medium. Suspension-adapted cellsare cryopreserved in medium with 10% DMSO.

Recombinant CHO cell lines stably expressing secreted activatable IL-2proteins are generated by transfection of suspension-adapted cells.During selection with the antibiotic Hygromycin B viable cell densitiesare measured twice a week, and cells are centrifuged and resuspended infresh selection medium at a maximal density of 0.1×10⁶ viable cells/mL.Cell pools stably expressing activatable IL-2 proteins are recoveredafter 2-3 weeks of selection at which point cells are transferred tostandard culture medium in shake flasks. Expression of recombinantsecreted proteins is confirmed by performing protein gel electrophoresisor flow cytometry. Stable cell pools are cryopreserved in DMSOcontaining medium.

Activatable IL-2 proteins are produced in 10-day fed-batch cultures ofstably transfected CHO cell lines by secretion into the cell culturesupernatant. Cell culture supernatants are harvested after 10 days atculture viabilities of typically >75%. Samples are collected from theproduction cultures every other day and cell density and viability areassessed. On day of harvest, cell culture supernatants are cleared bycentrifugation and vacuum filtration before further use.

Protein expression titers and product integrity in cell culturesupernatants are analyzed by SDS-PAGE.

Purification of Activatable IL-2 Proteins

Activatable IL-2 proteins are purified from CHO cell culturesupernatants in a two-step procedure. The constructs are subjected toaffinity chromatography in a first step followed by preparative sizeexclusion chromatography (SEC) on Superdex 200 in a second step. Samplesare buffer-exchanged and concentrated by ultrafiltration to a typicalconcentration of >1 mg/mL. Purity and homogeneity (typically >90%) offinal samples are assessed by SDS PAGE under reducing and non-reducingconditions, followed by immunoblotting using an anti-HSA or antiidiotype antibody as well as by analytical SEC, respectively. Purifiedproteins are stored at aliquots at −80° C. until use.

Example 7: Determination of Antigen Affinity by Flow Cytometry

The activatable IL-2 proteins are tested for their binding affinities tohuman CD20⁺ cells and cynomolgus CD20⁺ cells.

CD20⁺ cells are incubated with 100 μL of serial dilutions of theactivatable IL-2 proteins and at least one protease. After washing threetimes with FACS buffer the cells are incubated with 0.1 mL of 10 μg/mLmouse monoclonal anti-idiotype antibody in the same buffer for 45 min onice. After a second washing cycle, the cells are incubated with 0.1 mLof 15 μg/mL FITC-conjugated goat anti-mouse IgG antibodies under thesame conditions as before. As a control, cells are incubated with theanti-His IgG followed by the FITC-conjugated goat anti-mouse IgGantibodies without the activatable IL-2 proteins. The cells were thenwashed again and resuspended in 0.2 mL of FACS buffer containing 2 μg/mLpropidium iodide (PI) in order to exclude dead cells. The fluorescenceof 1×10⁴ living cells is measured using a Beckman-Coulter FC500 MPL flowcytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) ora Millipore Guava EasyCyte flow cytometer using the Incyte software(Merck Millipore, Schwalbach, Germany). Mean fluorescence intensities ofthe cell samples are calculated using CXP software (Beckman-Coulter,Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach,Germany). After subtracting the fluorescence intensity values of thecells stained with the secondary and tertiary reagents alone the valuesare then used for calculation of the K_(D) values with the equation forone-site binding (hyperbola) of the GraphPad Prism (version 6.00 forWindows, GraphPad Software, La Jolla Calif. USA).

CD20 binding and crossreactivity are assessed on the human CD20⁺ tumorcell lines. The K_(D) ratio of crossreactivity is calculated using theK_(D) values determined on the CHO cell lines expressing eitherrecombinant human or recombinant cynomolgus antigens.

Example 8: Cytotoxicity Assay

The activatable IL-2 protein is evaluated in vitro on its mediation ofimmune response to CD20⁺ target cells.

Fluorescence labeled CD20⁺ REC-1 cells (a Mantle cell lymphoma cellline, ATCC CRL-3004) are incubated with isolated PBMC of random donorsor CB15 T-cells (standardized T-cell line) as effector cells in thepresence of the activatable IL-2 protein and at least one protease.After incubation for 4 h at 37° C. in a humidified incubator, therelease of the fluorescent dye from the target cells into thesupernatant is determined in a spectrofluorimeter. Target cellsincubated without the activatable IL-2 protein and target cells totallylysed by the addition of saponin at the end of the incubation serve asnegative and positive controls, respectively.

Based on the measured remaining living target cells, the percentage ofspecific cell lysis is calculated according to the following formula:[1−(number of living targets_((sample))/number of livingtargets_((spontaneous)))]×100%. Sigmoidal dose response curves and EC₅₀values are calculated by non-linear regression/4-parameter logistic fitusing the GraphPad Software. The lysis values obtained for a givenantibody concentration are used to calculate sigmoidal dose-responsecurves by 4 parameter logistic fit analysis using the Prism software.

Example 9: Pharmacokinetics of Activatable IL-2 Proteins

The activatable IL-2 protein is evaluated for half-time elimination inanimal studies.

The activatable IL-2 protein is administered to cynomolgus monkeys as a0.5 mg/kg bolus injection into the saphenous vein. Another cynomolgusmonkey group receives a comparable IL-2 construct in size, but lacking aserum half-life extension element. A third and fourth group receive anIL-2 construct with serum half-life extension element and a cytokinewith CD20 and serum half-life extension elements respectively, and bothcomparable in size to the activatable IL-2 protein. Each test groupconsists of 5 monkeys. Serum samples are taken at indicated time points,serially diluted, and the concentration of the proteins is determinedusing a binding ELISA to CD20.

Pharmacokinetic analysis is performed using the test article plasmaconcentrations. Group mean plasma data for each test article conforms toa multi-exponential profile when plotted against the time post-dosing.The data are fit by a standard two-compartment model with bolus inputand first-order rate constants for distribution and elimination phases.The general equation for the best fit of the data for i.v.administration is: c(t)=Ae^(−60 t)+Be^(−βt), where c(t) is the plasmaconcentration at time t, A and B are intercepts on the Y-axis, and α andβ are the apparent first-order rate constants for the distribution andelimination phases, respectively. The α-phase is the initial phase ofthe clearance and reflects distribution of the protein into allextracellular fluid of the animal, whereas the second or β-phase portionof the decay curve represents true plasma clearance. Methods for fittingsuch equations are well known in the art. For example,A=D/V(α−k21)/(α−β), B=D/V(β−k21)/(α−β), and α and β (for α>β) are rootsof the quadratic equation: r²+(k12+k21+k10)r+k21k10=0 using estimatedparameters of V=volume of distribution, k10=elimination rate,k12=transfer rate from compartment 1 to compartment 2 and k21=transferrate from compartment 2 to compartment 1, and D=the administered dose.

Data analysis: Graphs of concentration versus time profiles are madeusing KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. SynergySoftware. Reading, Pa.). Values reported as less than reportable (LTR)are not included in the PK analysis and are not represented graphically.Pharmacokinetic parameters are determined by compartmental analysisusing WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.).Pharmacokinetic parameters are computed as described in Ritschel W A andKearns G L, 1999, IN: Handbook Of Basic Pharmacokinetics IncludingClinical Applications, 5th edition, American Pharmaceutical Assoc.,Washington, D.C.

It is expected that the activatable IL-2 protein has improvedpharmacokinetic parameters such as an increase in elimination half-timeas compared to proteins lacking a serum half-life extension element.

Example 10: Xenograft Tumor Model

The activatable IL-2 protein is evaluated in a xenograft model.

Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy)and subcutaneously inoculated with 4×10⁶ Ramos RA1 cells into the rightdorsal flank. When tumors reach 100 to 200 mm³, animals are allocatedinto 3 treatment groups. Groups 2 and 3 (8 animals each) areintraperitoneally injected with 1.5×10⁷ activated human T-cells. Threedays later, animals from Group 3 are subsequently treated with a totalof 9 intravenous doses of 50 μg activatable IL-2 protein (qd×9d). Groups1 and 2 are only treated with vehicle. Body weight and tumor volume aredetermined for 30 days.

It is expected that animals treated with the activatable IL-2 proteinhave a statistically significant delay in tumor growth in comparison tothe respective vehicle-treated control group.

Example 11: HEK Blue Assay

HEK-Blue IL-2 cells (InvivoGen) were plated in suspension at aconcentration of 50,000 cells/well in culture media with or without15-40 mg/ml human serum albumin (HSA) and stimulated with a dilutionseries of recombinant hIL-2 or activatable hIL-2 for 24 hours at 37° C.and 5% CO₂. Activity of uncleaved and cleaved activatable hIL-2 wastested. Cleaved inducible hIL-2 was generated by incubation with activeMMP9. IL-2 activity was assessed by quantification of Secreted AlkalinePhosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), acolorimetric based assay. Results are shown in FIGS. 11a, 11c, 11e and11 f.

Example 12: MC38 Experiments

The MC38 cell line, a rapidly growing colon adenocarcinoma cell linethat expresses MMP9 in vitro, was used. Using this tumor model, theability of fusion proteins to affect tumor growth was examined.

Example 12a: MC38 IL-2 Fusion Protein Treatment

Agents and Treatment:

Formulation Gr. N Agent dose Route Schedule   1^(#) 10 Vehicle — ip biwkx 2  2 7 ACP16 700 μg/animal ip biwk x 2  3 7 ACP16 230 μg/animal ipbiwk x 2  4 7 ACP16 70 μg/animal ip biwk x 2  5 7 ACP16 55 μg/animal ipbiwk x 2  6 7 ACP16 17 μg/animal ip biwk x 2  7 7 ACP132 361 μg/animalip biwk x 2  8 7 ACP132 119 μg/animal ip biwk x 2  9 7 ACP132 36μg/animal ip biwk x 2 10 7 ACP132 28 μg/animal ip biwk x 2 11 7 ACP132 9μg/animal ip biwk x 2 12 7 ACP21 540 μg/animal ip biwk x 2 13 7 ACP21177 μg/animal ip biwk x 2 14 7 ACP21 54 μg/animal ip biwk x 2 15 7 ACP2142 μg/animal ip biwk x 2 16 7 ACP21 13 μg/animal ip biwk x 2 17 7 ACP133210 μg/animal ip bid x 5 then 2-day pause then bid x 5 then 2-day pause18 7 ACP133 105 μg/animal ip bid x 5 then 2-day pause then bid x 5 then2-day pause 19 7 ACP133 40 μg/animal ip bid x 5 then 2-day pause thenbid x 5 then 2-day pause 20 7 ACP133 3 μg/animal ip bid x 5 then 2-daypause then bid x 5 then 2-day pause ^(#)Control Group

Results are shown in FIG. 17a-17m . The results show efficacy in tumorgrowth inhibition (TGI) with fusion protein treatments. Completeresponses (CR) were observed in ACP16 groups 55 μg/animal (FIG. 17c ),70 μg/animal (FIG. 17d ), and 230 μg/ml (FIG. 17e ). Addition of anequivalent IL-2 molar amounts to ACP16 using ACP132 (IL-2 with half-lifeextension element and without a blocker) showed high toxicity in allgroups except for the lowest dose, demonstrating the need for a blocker(FIGS. 17f-17i ). Additionally, ACP21, a construct with the blocker onlyand no half-life extension element, was ineffective at equivalent dosesto ACP16 (FIGS. 17j-17m ). The data demonstrates the need for a blockerand half-life extension element in the design of an effective IL-2fusion protein.

Example 12b: MC38 IL-2 Fusion Protein Treatment

Agents and Treatment:

Formulation Gr. N Agent dose Route Schedule   1^(#) 12 Vehicle — ip biwkx 2  2 8 ACP16 4.4 μg/animal ip biwk x 2  3 8 ACP16 17 μg/animal ip biwkx 2  4 8 ACP16 70 μg/animal ip biwk x 2  5 8 ACP16 232 μg/animal ip biwkx 2  6 8 ACP130 19 μg/animal ip biwk x 2  7 8 ACP130 45 μg/animal ipbiwk x 2  8 8 ACP130 180 μg/animal ip biwk x 2  9 8 ACP130 600 μg/animalip biwk x 1 12 8 ACP124 17 μg/animal ip biwk x 2 13 8 ACP124 70μg/animal ip biwk x 2 14 8 ACP124 230 μg/animal ip biwk x 2 15 8 ACP124700 μg/animal ip biwk x 2 16 8 IL-2-WTI 12 μg/animal ip bid x 5 then2-day pause then bid x 5 then 2-day pause 17 8 IL-2-WTI 36 μg/animal ipbid x 5 then 2-day pause then bid x 5 then 2-day pause ^(#)Control Group

Results are shown in FIGS. 13, 14, and 16. The results show efficacy intumor growth inhibition (TGI) with fusion protein treatments. Dosingwith ACP16 at 70 μg/animal and 232 μg/animal showed TGI efficacy (FIG.13a ). Equivalent doses of a non-cleavable version of ACP16 (designatedas ACP124) showed lack of TGI efficacy, demonstrating that a cleavablelinker may be required for in vivo efficacy (FIGS. 13b and 13c ).

Example 12c: Procedure for MC38 Experiments with Fusion ProteinTreatment

Mice were anaesthetized with isoflurane for implant of cells to reducethe ulcerations. CR female C57BL/6 mice were set up with 5×10⁵ MC38tumor cells in 0% Matrigel sc in flank. Cell Injection Volume was 0.1mL/mouse. Mouse age at start date was 8 to 12 weeks. Pair matches wereperformed when tumors reach an average size of 100-150 mm³ and begintreatment. Animals were treated with agents and doses as indicated forExamples 12a and 12b above. Body weights were taken at initiation andthen biweekly to the end. Caliper measurements were taken biweekly tothe end. Any adverse reactions were to be reported immediately. Anyindividual animal with a single observation of >than 30% body weightloss or three consecutive measurements of >25% body weight loss waseuthanized. Any group with a mean body weight loss of >20% or >10%mortality stopped dosing; the group was not euthanized and recovery isallowed. Within a group with >20% weight loss, individuals hitting theindividual body weight loss endpoint were euthanized. If the grouptreatment related body weight loss is recovered to within 10% of theoriginal weights, dosing resumed at a lower dose or less frequent dosingschedule. Exceptions to non-treatment body weight % recovery wereallowed on a case-by-case basis. Endpoint was tumor growth delay (TGD).Animals were monitored individually. The endpoint of the experiment wasa tumor volume of 1500 mm³ or 45 days, whichever comes first. Responderswere followed longer. When the endpoint was reached, the animals are tobe euthanized.

Example 12c: MC38 Re-Challenge

Cured mice (ACP16-treated) from Example 12b were re-challenged withtumor implantation 60 days after initial inoculation with MC38 tumorcells to determine whether anti-tumor memory had been established fromthe initial treatments.

Agents and Treatment:

Gr. N Agent Formulation dose Route Schedule  1^(#) 33 No — — — Treatment2 7 ACP16 70 μg/animal ip (ACP16 biwkx2) 3 8 ACP16 232 μg/animal  ip(ACP16 biwkx2) 5 5 IL-2-WTI 12 μg/animal ip (IL-2-WTI bid x 5 then 2-daypause then bid x 5 then 2-day pause) 6 7 IL-2-WTI 36 μg/animal ip(IL-2-WTI bid x 5 then 2-day pause then bid x 5 then 2-day pause)^(#)Control GroupProcedures:

Mice were anaesthetized with isoflurane for implant of cells to reducethe ulcerations. This portion of the study began on the day of implant(Day 1). Group 1 consisted of 33 CR female C57BL/6 mice set up with5×10⁵ MC38 tumor cells in 0% Matrigel subcutaneously in the flank.Groups 2-6 consisted of 33 CR female C57BL/6 mice set up with 5×10⁵ MC38tumor cells in 0% Matrigel sc in the left flank. The tumors from theprevious MC38 experiment (Example 12b) were implanted in the right flankof each animal. Cell Injection Volume was 0.1 mL/mouse. Age of controlmice at initiation was 14 to 17 weeks. These mice were age matched tomice from the previous MC38 experiment (Example 12b). No dosing ofactive agent occurred during re-challenge. Body Weights were takebiweekly until end, as were caliper measurements. Any adverse reactionsor death were reported immediately. Any individual animal with a singleobservation of >than 30% body weight loss or three consecutivemeasurements of >25% body weight loss was euthanized. Endpoint was tumorgrowth delay (TGD). Animals were monitored individually. The endpoint ofthe experiment was a tumor volume of 1000 mm³ or 45 days, whichevercomes first. Responders were followed longer when possible. When theendpoint was reached, the animals were euthanized.

All animals treated with ACP16 demonstrated development of immunologicalmemory against the tumor as they did not develop any tumors uponre-challenge, while naïve C57B16 control animals developed tumors at anormal rate. Results are shown in FIG. 15.

Example 13. Conditionally Active Fusion Proteins that Contain a BlockingMoiety that is a Serum Albumin Binding Domain

This example describes the production and activity of fusion proteins,preferably cytokines, that have inducible activity, i.e., they areinactive until induced, typically by separation of a blocking moietyfrom the active moiety upon cleavage of a linker between the blockingmoiety and the active moiety. The fusion proteins contain a singleantibody variable domain (a dAb) that binds serum albumin via the CDRloops, and binds to an active moiety (here an anti-CD3 scFV) via one ormore non-CDR loops (e.g., the C loop). The serum albumin-bindingblocking moiety is operably linked to the active moiety through aprotease-cleavable linker, and active moiety is operably linked to atargeting domain (here an anti-epidermal growth factor receptor (EGFR)dAb or anti-prostate-specific membrane antigen (PSMA) dAb) through alinker that is not protease cleavable. These fusion proteins can beadministered as inactive proteins that become activated upon cleavage ofthe protease-cleavable linker and subsequent release of the inhibitoryalbumin-binding domain. The anti-CD3 scFV in the fusion proteins is asurrogate for a desired cytokine in the fusion proteins described inthis disclosure. Similar fusion proteins that contain a desired cytokine(e.g., IL-2, IL-12, an Interferon) or functional fragment or muteinthereof, a targeting domain and an albumin-binding dAb that also bindsand inhibits the cytokine or functional fragment or mutein thereof canbe prepared using the methods described and exemplified herein.Anti-serum albumin dAb that bind and inhibit the activity of a desiredcytokine or functional fragment or mutein thereof can provide bothsteric masking of the cytokine (through the cytokines proximity to boundserum albumin) and specific masking of the cytokine (through binding tocytokine via the non-CDR loop (e.g., the C loop)). Anti-serum albumindAb that bind and inhibit the activity of a desired cytokine orfunctional fragment or mutein thereof can be obtained using suitablemethods, such as by introducing amino acid sequence diversity into thenon-CDR loops (e.g., C loop) of an anti-serum albumin binding dAb andscreening for binding to the desired cytokine. Any suitable methods canbe used for the selection, such as phage display. For example, anexemplary anti-serum albumin dab that can be used has the followingsequence, and the amino acid sequence in the C loop (Bold Underlined)can be diversified (e.g., randomized) and resulting dAbs screened forbinding to serum albumin via CDR interaction and to cytokine via non-CDRloop interaction. If desired, the amino acid sequence of a knowncytokine binding peptide can be grafted into the C loop.

(SEQ ID NO: 137) EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQ GGGGGLDGNEE PGGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSVSSQGTLVTVSSA. Protease Activation of ProTriTAC Leads to Significantly EnhancedActivity In Vitro

Purified ProTriTAC (prodrug), non-cleavable ProTriTAC [prodrug(non-cleavable)], and recombinant active drug fragment mimicking theprotease-activated ProTriTAC (active drug) were tested for binding torecombinant human CD3 in an ELISA assay, binding to purified humanprimary T cells in a flow cytometry assay, and functional potency in a Tcell-dependent cellular cytotoxicity assay.

For ELISA, soluble ProTriTAC proteins at the indication concentrationswere incubated with immobilized recombinant human CD3e (R&D Systems) for1 h at room temperature in PBS supplemented with 15 mg/ml human serumalbumin. Plates were blocked using SuperBlock (Thermo Fisher), washedusing PBS with 0.05% Tween-20, and detected using a non-competitiveanti-CD3 idiotype monoclonal antibody 11D3 followed byperoxidase-labeled secondary antibody and TMB-ELISA substrate solution(Thermo Fisher).

For flow cytometry, soluble ProTriTAC proteins at the indicatedconcentrations were incubated with purified human primary T cells for 1h at 4° C. in the presence of PBS with 2% fetal bovine serum and 15mg/ml human serum albumin. Plates were washed with PBS with 2% fetalbovine serum, detected using AlexaFluor 647-labeled non-competitiveanti-CD3 idiotype monoclonal antibody 11D3, and data was analyzed usingFlowJo 10 (FlowJo, LLC).

For functional potency in a T cell-dependent cellular cytotoxicityassays, soluble ProTriTAC proteins at the indicated concentrations wereincubated with purified resting human T cells (effector cell) and HCT116cancer cell (target cell) at 10:1 effector:target cell ratio for 48 h at37° C. The HCT116 target cell line has been stably transfected with aluciferase reporter gene to allow specific T cell-mediated cell killingmeasurement by ONE-Glo (Promega).

B. ProTriTAC Exhibits Potent, Protease-Dependent, Anti-Tumor Activity ina Rodent Tumor Xenograft Model

ProTriTAC was evaluated for their anti-tumor activity in vivo in anHCT116 subcutaneous xenograft tumor admixed with expanded human T cellsin immunocompromised NCG mice. Specifically, 5×106 HCT116 cells wereadmixed with 2.5×106 expanded T cells per mouse on day 0. Dosing ofProTriTACs were performed starting on the following day with a q.d.×10schedule via intraperitoneal injection. Tumor volume measurements weredetermined using caliper measurements and calculated using the formulaV=(length×width×width)/2 at the indicated times.

C. Expression, Purification and Stability of Exemplary ProTriTACTrispecific Molecules Protein Production

Sequences encoding inducible fusion protein molecules were cloned intomammalian expression vector pcDNA 3.4 (Invitrogen) preceded by a leadersequence and followed by a 6×Histidine Tag (SEQ ID NO: 136). Expi293Fcells (Life Technologies A14527) were maintained in suspension inOptimum Growth Flasks (Thomson) between 0.2 to 8×1e6 cells/ml in Expi293 media. Purified plasmid DNA was transfected into Expi293 cells inaccordance with Expi293 Expression System Kit (Life Technologies,A14635) protocols, and maintained for 4-6 days post transfection.Alternatively sequences encoding the fusion protein molecules werecloned into mammalian expression vector pDEF38 (CMC ICOS) transfectedinto CHO-DG44 dhfr-cells, stable pools generated, and cultured inproduction media for up to 12 days prior to purification. The amount ofthe exemplary fusion proteins in conditioned media was quantified usingan Octet RED 96 instrument with Protein A tips (ForteBio/Pall) using acontrol fusion protein for a standard curve. Conditioned media fromeither host cell was filtered and partially purified by affinity anddesalting chromatography. Fusion proteins were subsequently polished byion exchange and upon fraction pooling formulated in a neutral buffercontaining excipients. Final purity was assessed by SDS-PAGE andanalytical SEC using an Acquity BEH SEC 200 1.7u 4.6×150 mm column(Waters Corporation) resolved in an aqueous/organic mobile phase withexcipients at neutral pH on a 1290 LC system and peaks integrated withChemstation CDS software (Agilent). Fusion proteins purified from CHOhost cells are shown in the SDS-PAGE depicted below.

Stability Assessment

Purified fusion proteins in two formulations were sub-aliquoted intosterile tubes and stressed by five freeze-thaw cycles each comprisinggreater than 1 hour at −80° C. and room temperature or by incubation at37° C. for 1 week. Stressed samples were evaluated for concentration andturbidity by UV spectrometry using UV transparent 96 well plates(Corning 3635) with a SpectraMax M2 and SoftMaxPro Software (MolecularDevices), SDS-PAGE, and analytical SEC and compared to the same analysisof control non-stressed samples. An overlay of chromatograms fromanalytical SEC of control and stressed samples for a single exemplaryProTriTAC molecule purified from 293 host cells is depicted below.

The results show that ProTriTACs were produced in comparable yields toregular TriTACs from CHO stable pools; and that the proteins were stableafter repeated freeze-thaws and 37° C. for 1 week.

D. Demonstration of Functional Masking and Stability of ProTriTAC InVivo in a Three-Week Cynomolgus Monkey Pharmacokinetic Study

Single dose of PSMA-targeting ProTriTAC (SEQ ID NO: 119), non-cleavableProTriTAC (SEQ ID NO: 120), non-masked/non-cleavable TriTAC (SEQ ID NO:123), and active drug mimicking protease-activated ProTriTAC (SEQ ID NO:121) was dosed into cynomolgus monkeys at 0.1 mg/kg via intravenousinjection. Plasma samples were collected at the indicated time points.ProTriTAC concentrations were determined using ligand binding assayswith biotinylated recombinant human PSMA (R&D systems) and sulfo-taggedanti-CD3 idiotype antibody cloned 11D3 in a MSD assay (Meso ScaleDiagnostic, LLC). Pharmacokinetic parameters were estimated usingPhoenix WinNonlin pharmacokinetic software using a non-compartmentalapproach consistent with the intravenous bolus route of administration.

To calculate the rate of in vivo prodrug conversion, the concentrationof active drug in circulation was estimated by solving the followingsystem of differential equations where P is the concentration ofprodrug, A is the concentration of active drug, k_(a) is the rate ofprodrug activation in circulation, k_(c,P) is the clearance rate of theprodrug, and k_(c,A) is the clearance rate of the active drug.

$\frac{dP}{dt} = {{- k_{c,P}}P}$$\frac{dA}{dt} = {{k_{a}P} - {k_{c,A}A}}$

The clearance rates of the prodrug, active drug, and a non-cleavableprodrug control (κ_(c,NCLV)) were determined empirically in cynomolgusmonkeys. To estimate the rate of prodrug activation in circulation, weassumed that the difference between the clearance rate of cleavableprodrug and non-cleavable prodrug arose solely from non-specificactivation in circulation. Therefore, the rate of prodrug conversion toactive drug in circulation was estimated by subtracting the clearancerate of the cleavable prodrug from the non-cleavable prodrug.k _(a) =k _(c,NCLV) −k _(c,P)

The initial concentration of prodrug in circulation was determinedempirically and the initial concentration of active drug was assumed tobe zero.

Results and Discussion

The results of Example 13 show that fusion proteins that contain apolypeptide with desired therapeutic activity, such as a cytokine orfunctional fragment or mutein thereof or anti-CD3 scFV, can be preparedin which the therapeutic activity is masked by a masking domain thatbinds to both serum albumin and to the active polypeptide. The maskingdomain is operably linked to the active domain through aprotease-cleavable linker. The results show that this type of fusionprotein can be administered as an inactive protein that becomesactivated upon protease cleavage at the desired location of therapeuticactivity, such as, at a tumor.

Amino acid sequences of fusion proteins used in Example 13 are given SEQID NO: 116-123.

Sample fusion protein constructs are detailed in Table 3. In Table 3,“L” is an abbreviation of “linker”, “cleav. link.” and “LX” areabbreviations of different cleavable linkers, and “HSA” indicates humanserum albumin (HSA).

TABLE 3 CONSTRUCT PERMUTATION TABLE Construct Name Construct DescriptionACP63 anti-FN CGS-2 scFv (Vh/Vl)-6xHis ACP12 (anti-EpCAM)-IL2-(cleav.link.)-(anti-HSA)-blocker-6xHis ACP13(anti-EpCAM)-Blocker2-(anti-HSA)-(cleav. link.)-IL2-6xHis ACP14Blocker2-Linker-(cleav. link.)-IL2-(cleav. link.)-(anti-HSA)-6xHis ACP15Blocker2-Linker-(anti-HSA)-Linker-(cleav. link.)- IL2 -6xHis ACP16IL2-(cleav. link.)-(anti-HSA)-Linker-(cleav. link.)-Blocker2-6xHis ACP17(anti-EpCAM)-Linker-IL2-(cleav. link.)-(anti-HSA)-Linker-(cleav.link.)-Blocker2-6xHis ACP18 (anti-EpCAM)-Linker-IL2-(cleav.link.)-(anti-HSA)-Linker-vh(cleav. link.)vl-6xHis ACP19 IL2-(cleav.link.)-Linker-Blocker2-Linker-(anti-HSA)-Linker-(anti-EpCAM) -6xHisACP20 IL2-(cleav. link.)-Blocker2-6xHis ACP21 IL2-(cleav.link.)-Linker-Blocker2-6xHis ACP22 IL2-(cleav.link.)-Linker-blocker-(cleav.link.)-(anti-HSA)-Linker-(anti-EpCAM)-6xHis ACP23 (anti-FOLR1)-(cleav.link.)-Blocker2-Linker-(cleav. link.)-(anti-HSA)-(cleav.link.)-IL2-6xHis ACP24 (Blocker2)-(cleav. link.)-(IL2)-6xHis ACP25Blocker2-Linker-(cleav. link.)-IL2-6xHis ACP26(anti-EpCAM)-Linker-IL2-(cleav. link.)-(anti-HSA)-Linker-blocker(NARA1Vh/Vl) ACP27 (anti-EpCAM)-Linker-IL2-(cleav.link.)-(anti-HSA)-Linker-blocker(NARA1 Vl/Vh) ACP28 IL2-(cleav.link.)-Linker-Blocker2-(NARA1Vh/Vl)-Linker-(anti-HSA)-Linker-(anti-EpCAM) ACP29 IL2-(cleav.link.)-Linker-Blocker2-(NARA1Vl/Vh)-Linker-(anti-HSA)-Linker-(anti-EpCAM) ACP38 IL2-(cleav.link.)-blocker-(anti-HSA)-(anti-EpCAM)-6xHis ACP39 (anti-EpCAM)-(cleav.link.)-(anti-HSA)-(cleav. link.)-Blocker2-(cleav. link.)-IL-2-6xHisACP40 CD25ecd-Linker-(cleav. link.)-IL2-6xHis ACP41 IL2-(cleav.link.)-Linker-CD25ecd-6xHis ACP42(anti-HSA)-Linker-CD25ecd-Linker-(cleav. link.)-IL2-6xHis ACP43IL2-(cleav. link.)-Linker-CD25ecd-Linker-(anti-HSA)-6xHis ACP44IL2-(cleav. link.)-Linker-CD25ecd-(cleav. link.)-(anti-HSA)-6xHis ACP45(anti-HSA)-(cleav. link.)-Blocker2-Linker-(cleav. link.)-IL2-6xHis ACP46IL2-(cleav. link.)-linker-vh(cleav.link.)vl-Linker-(anti-HSA)-L-(anti-EpCAM)-6xHis ACP47(anti-EpCAM)-Linker-IL2-(CleavableLinker)-(anti-HSA)-Linker-Blocker2-6xHis ACP48 IL2-(cleav.link.)-Blocker2-Linker-(anti-HSA)-6xHis ACP49 IL2-(cleav.link.)-Linker-Blocker2-Linker-(anti-HSA)-6xHis ACP92 (anti-HSA)-(16mercleav. link.)-IL2-(16mer cleav. Link.)-(anti-HSA)-6XHis ACP93(anti-EpCAM)-(anti-HSA)-(anti-EpCAM)-Blocker2-(cleav. link.)-IL2-6xHisACP94 (anti-EpCAM)-(anti-HSA)-Blocker2-(cleav. link.)-IL2-6xHis ACP95(anti-EpCAM)-(anti-HSA)-(cleav. link.)-IL2-6xHis ACP96(anti-EpCAM)-(16mer cleav. link.)-IL2-(16mer cleav. link.)-(anti-HSA)ACP97 (anti-EpCAM)-(anti-HSA)-(cleav. link.)-IL2-(cleav.link.)-(anti-HSA)-6xHis ACP99 (anti-EpCAM)-Linker-IL2-(cleav.link.)-(anti-HSA)-6xHis ACP100 (anti-EpCAM)-Linker-IL2-6xHis ACP101IL2-(cleav. link.)-(anti-HSA)-6xHis ACP102 (anti-EpCAM)-(cleav.link.)-IL2-(cleav. link.)-(anti-HSA)-Linker-blocker-6xHis ACP103IL2-(cleav.link.)-Linker-Blocker2-Linker-(anti-HSA)-Linker-(antiI-FOLR1)-6xHisACP104 (anti-FOLR1)-IL2-(cleav. link.)-(anti-HSA)-Linker-Blocker2-6xHisACP105 Blocker2-Linker-(cleav. link.)-IL2-(cleav.link.)-(anti-HSA)-Linker-(anti-FOLR1)-6xHis ACP106(anti-FOLR1)-Linker-(anti-HSA)-(cleav. link.)-blocker-Linker-(cleav.link.)-IL2 -6xHis ACP107 Blocker2-Linker-(anti-HSA)-(cleav.link.)-IL2-Linker-(anti-FOLR1)-6xHis ACP108 (anti-EpCAM)-IL2-(Duallycleav. link.)-(anti-HSA)-Linker-blocker-6xHis ACP117 anti-FN CGS-2 scFv(Vh/Vl)-6xHis ACP118 NARA1 Vh/Vl non-cleavable ACP119 NARA1 Vh/Vlcleavable ACP120 NARA1 Vl/Vh non-cleavable ACP121 NARA1 Vl/Vh cleavableACP124IL2-Linker-(anti-HSA)-Linker-Linker-blocker_(non-cleavable_control)ACP132 IL2-L-HSA ACP141 IL2-L-hAlb ACP142 IL2-(cleav. link.)-hAlb ACP144IL2-(cleav. link.)-HSA-LX-blocker-L-FOLR1 ACP145 FOLR1-L-IL2-(cleav.link.)-HSA-LX-blocker ACP146 FOLR1-(cleav. link.)-IL2-(cleav.link.)-HSA-LX-blocker ACP133 IL-2-6x His ACP147 IL2-(cleav.link.)-HSA-LX-blocker-L-TAA ACP148 TAA-L-IL2-(cleav.link.)-HSA-LX-blocker ACP149 TAA-(cleav. link.)-IL2-(cleav.link.)-HSA-LX-blocker ACP153 IL2-(cleav. link.)-(anti-HSA)-linker(cleav.link.)-Blocker2 ACP154 IL2-(cleav. link.)-(anti-HSA)-linker(cleav.link.)-Blocker2 ACP155 IL2-(cleav. link.)-(anti-HSA)-linker(cleav.link.)-Blocker2 ACP156 IL2-(cleav. link.)-(anti-HSA)-linker(cleav.link.)-Blocker2 ACP157 IL2-(cleav. link.)-(anti-HSA)-linker(cleav.link.)-Blocker2

TABLE 4 SEQUENCE TABLE SEQ ID NO. Name Sequence 1 HumanMYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD IL-2LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSETTFMCEYADE TATIVEFLNR WITFCQSIISTLT 2 HumanMKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGE serumENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD albuminESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEPERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLKKYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ GLKCASLQKF GERAFKAWAVARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADDRADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVENDEMPADLPSLA ADFVGSKDVC KNYAEAKDVF LGMFLYEYARRHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDEFKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVPQVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDCLSVF LNQLCVLHEK TPVSDRVTKCCTESLVNGRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKK QTALVELVKHK PKATKEQLKAVMDDFAAFVEKCCKADDKET CFAEEGKKLVAASQAALGL 45 ACP12QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL (IL-2VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY fusionCNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil protein)nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 46 ACP13QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL (IL-2VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY fusionCNALYGTDYWGKGTQVTVSSggggsggggsggggsEVQLVESGGGLVQPG protein)GSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 47 ACP14EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEW (IL-2VAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYY fusionCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQS protein)PSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 48 ACP15EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEW (IL-2VAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYY fusionCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQS protein)PSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 49 ACP16aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaq(IL-2sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGfusion LPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGK protein)GLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 50 ACP17QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL (IL-2VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY fusionCNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil protein)nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGT KVEIKHHHHHH 51 ACP18QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL (IL-2VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY fusionCNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil protein)nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSsggpgpagmkglpgsDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 52 ACP19aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaq(IL-2sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGfusion LPGSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSC protein)AASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSHH HHHH** 53 ACP20aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaq(IL2sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGfusion LPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGK protein)GLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFG GGTKVEIKHHHHHH 54ACP21aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaq(IL-2sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGfusion LPGSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSC protein)AASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 55 ACP22aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaq(IL-2sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGfusion LPGSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSC protein)AASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTV SSHHHHHH 56 ACP23QVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQRE (IL-2FVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVY fusionVCNRNFDRIYWGQGTQVTVSSSGGPGPAGMKGLPGSEVQLVESGGG protein)LVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 57 ACP24EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEW (IL-2VAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYY fusionCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQS protein)PSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmlifkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 58 ACP25EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEW (IL-2VAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYY fusionCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQS protein)PSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 59 ACP26QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL (IL-2VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY fusionCNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehilldlqmil protein)nginnyknpldtrmlifkfympkkatelkhlqcleeelkpleevinlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSggggsggggsggggsDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKHHHHHHEPEA 60 ACP27QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL (IL-2VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY fusionCNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil protein)nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsggggsggggsQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSHHHHHHEPEA 61 ACP28aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaq(IL-2sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGfusion LPGSggggsggggsggggsggggsggggsQVQLQQSGAELVRPGTSVKVSCKAS protein)GYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSggggsggggsggggsDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSHH HHHHEPEA 62 ACP29aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaq(IL-2sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGfusion LPGSggggsggggsggggsggggsggggsDIVLTQSPASLAVSLGQRATISCKAS protein)QSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsggggsggggsQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSHHH HHHEPEA 63 IL-2Ra        10         20         30         40         50MDSYLLMWGL LTFIMVPGCQ AELCDDDPPE IPHATFKAMA YKEGTMLNCE        60         70         80         90        100CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE       110        120        130        140        150QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY       160        170        180        190        200QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ       210        220        230        240        250ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL       260        270 ISVILLSGLT WQRRQRKSRR TI 64 IL-2Rb        10         20         30         40         50MAAPALSWRL PLLILLLPLA TSWASAAVNG TSQFTCFYNS RANISCVWSQ        60         70         80         90        100DGALQDTSCQ VHAWPDRRRW NQTCELLPVS QASWACNLIL GAPDSQKLTT       110        120        130        140        150VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNI       160        170        180        190        200SWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWIELETLTP       210        220        230        240        250DTQYEFQVRV KPLQGEFTTW SPWSQPLAFR TKPAALGKDT IPWLGHLLVG       260        270        280        290        300LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFS QLSSEHGGDV       310        320        330        340        350QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS       360        370        380        390        400SNHSLTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP       410        420        430        440        450TGSSPQPLQP LSGEDDAYCT FPSRDDLLLF SPSLLGGPSP PSTAPGGSGA       460        470        480        490        500GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE LVLREAGEEV       510        520        530        540        550PDAGPREGVS FPWSRPPGQG EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V 65 IL-2Rg        10         20         30         40         50MLKPSLPFTS LLFLQLPLLG VGLNTTILTP NGNEDTTADF FLTTMPTDSL        60         70         80         90        100SVSTLPLPEV QCFVFNVEYM NCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ       110        120        130        140        150KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRR QATQMLKLQN       160        170        180        190        200LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQ       210        220        230        240        250SVDYRHKFSL PSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT       260        270        280        290        300SKENPFLFAL EAVVISVGSM GLIISLLCVY FWLERTMPRI PTLKNLEDLV       310        320        330        340        350TEYHGNESAW SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASP        360CNQHSPYWAP PCYTLKPET 66 ACP63mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTESSYA (Anti-FNMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLY CGS-2LQMNSLRAEDTAVYYCARGVGAFRPYRKHEWGQGTLVTVSRggggsg scFv)gggsggggsSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTTTGAQAEDEADYYCNSSPFEHNLVVFGGGTKLTVLHHHHHHEPEA 67 ACP38mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGETES protein)SYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTEGGGTKVEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTESKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSHHHHHH 68 ACP39mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGMKG protein)LPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH** 69 ACP40mdmrvpaqllgllllwligarcelcdddppeiphatfkamaykegtmlnceckrgfrriksgslymlctgn(IL-2sshsswdnqcqctssatrnttkqvtpqpeeqkerkttemqspmqpvdqaslpghcrepppweneaterifusionyhfvvgqmvyyqcvqgyralhrgpaesyckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpprotein)esetsclvtttdfqiqtemaatmetsiftteyqggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 70 ACP41mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggselcdddppeiphprotein)atfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrnttkqvtpqpeeqkerkttemqspmqpvdqaslpghcrepppweneateriyhfvvgqmvyyqcvqgyralhrgpaesvckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclvtttdfqiqtemaatmetsiftteyqHHHHHH 71 ACP42 mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG(IL-2 MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLY fusionLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggselc protein)dddppeiphatfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrnttkqvtpqpeeqkerkttemqspmqpvdqaslpghcrepppweneateriyhfvvgqmvyyqcvqgyralhrgpaesvckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclvtttdfqiqtemaatmetsiftteyqggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 72 ACP43mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggselcdddppeiphprotein)atfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrnttkqvtpqpeeqkerkttemqspmqpvdqaslpghcrepppweneateriyhfvvgqmvyyqcvqgyralhrgpaesvckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclvtttdfqiqtemaatmetsiftteyqggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 73 ACP44mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggselcdddppeiphprotein)atfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrnttkqvtpqpeeqkerkttemqspmqpvdqaslpghcrepppweneateriyhfvvgqmvyyqcvqgyralhrgpaesvckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclvtttdfqiqtemaatmetsiftteyqSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 74 ACP45mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG (IL-2MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLY fusionLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGL protein)PGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 75 ACP46mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggsEVQLVESGprotein) GGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSsggpgpagmkglpgsDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKG TQVTVSSHHHHHH 76 ACP47mdmrvpaqllgllllwirgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsggggsa protein)ptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEI KHHHHHH 77 ACP48mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFS protein)SYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGT LVTVSSHHHHHH 78 ACP49mdmrvpaqllgllllwligarcaptssstkktqlqlehllidlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggsEVQLVESGprotein) GGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 79 ACP92mdmrvpaqllgllllwirgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG (IL-2MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLY fusionLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGL protein)PGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 80 ACP93mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSgsgsgsgsgsgsgsgsE protein)VQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSgsgsgsgsgsgsgsgsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSgsgsgsgsgsgsgsgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 81 ACP94mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSgsgsgsgsgsgsgsgsE protein)VQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSgsgsgsgsgsgsgsgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 82ACP95 mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSgsgsgsgsgsgsgsgsE protein)VQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevknlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 83 ACP96mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGMKG protein)LPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 84 ACP97mdmrvpaqllgllllwirgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsggggsE protein)VQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpUtrmitflcfympkkatelkhlqcleeelkpleevinlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSV SSQGTLVTVSSHHHHHH 85ACP99 mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsggggsa protein)ptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 86 ACP100mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsggggsa protein)ptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 87ACP101mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFS protein)KFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 88 ACP102mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGMKG protein)LPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGG TKVEIKHHHHHH 89 ACP103mdmrypaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmitfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggsEVQLVESGprotein) GGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYVCNRNFDRI YWGQGTQVTVSSHHHHHH 90ACP104 mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSV (IL-2MAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYL fusionQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSaptssstkktqlqlehllldl protein)qmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 91 ACP105mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYT (IL-2LAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYL fusionQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGG protein)GSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYVCNR NFDRIYWGQGTQVTVSSHHHHHH92 ACP106 mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSV (IL-2MAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYL fusionQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggggsE protein)VQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 93 ACP107mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYT (IL-2LAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYL fusionQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGG protein)GSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistllggggsggggsggggsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVS SHHHHHH 94 ACP108mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsggggsa protein)ptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSrgetgpaaPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYT FGGGTKVEIKHHHHHH 95ACP117 mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYA (Anti-FNMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLY CGS-2LQMNSLRAEDTAVYYCARGVGAFRPYRKHEWGQGTLVTVSRggggsg scFv)gggsggggsSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTTTGAQAEDEADYYCNSSPFEHNLVVFGGGTKLTVLHHHHHHEPEA 96 ACP118mdmrvpaqllgllllwlrgarcQVQLQQSGAELVRPGTSVKVSCKASGYAFTNY (NARA1LIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTA Vh/V1YMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSggggs non-ggggsggggsDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWY cleavable)QQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKHHHHHHEPEA 97 ACP119mdmrvpaqllgllllwlrgarcQVQLQQSGAELVRPGTSVKVSCKASGYAFTNY (NARA1LIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTA Vh/V1YMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSSGG cleavable)PGPAGMKGLPGSDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKHHHHHHEPEA 98 ACP120mdmrvpaqllgllllwlrgarcDIVLTQSPASLAVSLGQRATISCKASQSVDYDG (NARA1DSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHP Vl/VhVEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsggggsggggsQVQLQQ non-SGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINP cleavable)GSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSHHHHHHEPEA 99 ACP121mdmrvpaqllgllllwlrgarcDIVLTQSPASLAVSLGQRATISCKASQSVDYDG (NARA1DSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHP Vl/VhVEEEDAATYYCQQSNEDPYTFGGGTKLEIKSGGPGPAGMKGLPGSQV cleavable)QLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSHHHHHHEPEA 100 ACP124mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGM protein)SWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHHEPEA 101 ACP132mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusionistltggggsggggsggggsdahksevahrfkdlgeenfkalvliafaqylqqcpfedhvklynevtefaktproteincvadesaencdkslhtlfgalctvatlretygemadccakqepernecflqhkddnpnlprlvrpevdvmctafhdneetflkkylyeiarrhpyfyapellffakrykaafteccqaadkaacllpkldelrdegkassakqrlkcaslqkfgerafkawavarlsqrfpkaefaevsklvtdltkvhtecchgdllecaddradlakyicenqdsissklkeccekpllekshciaevendempadlpslaadfveskdvcknyaeakdvflgmflyeyarrhpdysvvlllrlaktyettlekccaaadphecyakvfdefkplveepqnlikqncelfeqlgeykfqnallvrytkkvpqvstptlvevsrnlgkvgskcckhpeakrmpcaedylsvvlnqlcvlhektpysdrytkccteslvnrrpcfsalevdetyvpkefnaetftfhadictlsekerqikkqtalvelvkhkpkatkeqlkaymddfaafvekcckaddketcfaeegkklvaasqaalglHHHHHHEPEA 102 ACP141mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusionistltggggsggggsggggsdahksevahrfkdlgeenfkalvliafaqylqqcpfedhvklvnevtefaktprotein)cvadesaencdkslhtlfgdklctvatlretygemadccakqepernecflqhkddnpnlprlvrpevdvmctafhdneetflkkylyeiarrhpyfyapellffakrykaafteccqaadkaacllpkldelrdegkassakqrlkcaslqkfgerafkawavarlsqrfpkaefaevsklvtdltkvhtecchgdllecaddradlakyicenqdsissklkeccekpllekshciaevendempadlpslaadfveskdvcknyaeakdvflgmflyeyarrhpdysvvlllrlaktyettlekccaaadphecyakvfdefkplveepqnlikqncelfeqlgeykfqnallvrytkkvpqvstptlveysrnlgkvgskcckhpeakrmpcaedylsvvlnqlcvlhektpysdrvtkccteslvnrrpcfsalevdetyvpkefnaetftfhadictlsekerqikkqtalvelvkhkpkatkeqlkavmddfaafvekcckaddketcfaeegkklvaasqaalglHHHHHHEPEA 103 ACP142mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSdahksevahrfkdlgeenfkalvliafaqylqqcpfedhvklvneprotein)vtefaktcvadesaencdkslhtlfgdklctvatlretygemadccakqepernecflqhkddnpnlprlvrpevdvmctafhdneetflkkylyeiarrhpyfyapellffakrykaafteccqaadkaacllpkldelrdegkassakqrlkcaslqkfgerafkawavarlsqrfpkaefaevsklvtdltkvhtecchgdllecaddradlakyicenqdsissklkeccekpllekshciaevendempadlpslaadfveskdvcknyaeakdvflgmflyeyarrhpdysvvlllrlaktyettlekccaaadphecyakvfdefkplyeepqnlikqncelfeqlgeykfqnallvrytkkvpqvstptlvevsrnlgkvgskcckhpeakrmpcaedylsvvlnqlcvlhektpvsdrvtkccteslvrrpcfsalevdetyvpkefnaetftfhadictlsekerqikkqtalvelvkapkatkeqlkavmddfaafvekcckaddketcfaeegkklvaasqaalglHHHHHHEPEA 104 ACP144mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFS protein)KFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSHHHHHHEPEA 105 ACP145mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSV (IL-2MAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYL fusionQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggggsap protein)tssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 106 ACP146mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSV (IL-2MAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYL fusionQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSSGGPGPAGMKG protein)LPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 107 ACP133mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2-elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsi6xHis) istltHHHHHH (″6xHis″ disclosed as SEQ ID NO: 136) 108 ACP147mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsifusion istltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFS protein)KFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSHHHHHHEPEA 109 ACP148mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsggggsa protein)ptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 110 ACP149mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI (IL-2MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYL fusionQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGMKG protein)LPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 111 ACP153mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiConju- istltsggpGPAGLYAQpgsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF gate)GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggssggpGPAGLYAQpgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 112 ACP154mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiConju- istltsggpPGGPAGIGpgsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG gate)MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggssggpPGGPAGIGpgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 113 ACP155mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiConju- istltsggpALFKSSFPpgsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG gate)MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggssggpALFKSSFPpgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 114 ACP156mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiConju- istltsggpPLAQKLKSSpgsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF gate)GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggssggpPLAQKLKSSpgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 115 ACP157mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkat(IL-2elkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiConju- istltsggpPGGPAGIGalfkssfpPLAQKLKSSpgsEVQLVESGGGLVQPGNSL gate)RLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggssggpPGGPAGIGaltkssfpPLAQKLKSSpgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGG GTKVEIKHHHHHHEPEA 116EGFR EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQGGGGGLD (G8)GNEEPGGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMN ProdrugSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGKPLGLQARVVGG C1486GGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLTLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWASGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQ GTLVTVSSHHHHHH 117EGFR EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQGGGGGLD (G8) Non-GNEEPGGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMN cleavableSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGGSGGVVGG ProdrugGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQ C1756APRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLTLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWASGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQ GTLVTVSSHHHHHH 118EGFR VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQ (G8)QKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAE ActiveYYCTLWYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESG DrugGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSK C1300YNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLTLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWASGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAGYQINSGNYNFKDYE YDYWGQGTLVTVSSHHHHHH119 PSMA EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQGGGGGLD ProdrugGNEEPGGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMN C1872SLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGKPLGLQARVVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHVVVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSSHHHHHH 120 PSMAEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQGGGGGLD Non-GNEEPGGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMN cleavableSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGGSGGVVGG ProdrugGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQ C1873APRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHVVVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSSHHHHHH 121 PSMAVVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQ ActiveQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAE DrugYYCTLWYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESG C1875GGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSSH HHHHH 122 GFPQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVA TriTACGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVN C646VGFEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH 123 non-EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE masked/WVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV non-YYCTIGGSLSVSSQGTLVTVSSGGGGSGGGGSGGVVGGGGTQTVVTQ cleavableEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGT TriTACKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVF C1874GGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSSHHHHHH 124 Blocker 2mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYT (IL-2LAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYL blocker)QMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYT YPYTFGGGTKVEIKHHHHHH

INCORPORATION BY REFERENCE

The entire disclosures of all patent and non-patent publications citedherein are each incorporated by reference in their entireties for allpurposes.

OTHER EMBODIMENTS

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in this application, in applications claiming priority fromthis application, or in related applications. Such claims, whetherdirected to a different invention or to the same invention, and whetherbroader, narrower, equal, or different in scope in comparison to theoriginal claims, also are regarded as included within the subject matterof the inventions of the present disclosure.

The invention claimed is:
 1. A fusion polypeptide of the formula:[A]-[L1]-[B]-[L2]-[D] or [A]-[L1]-[D]-[L2]-[B] or [D]-[L2]-[B]-[L1]-[A]or [B]-[L2]-[D]-[L1]-[A] or [D]-[L1]-[B]-[L1]-[A] or[B]-[L1]-[D]-[L1]-[A] or [B]-[L1]-[A]-[L1]-[D] or [D]-[L1]-[A]-[L1]-[B],wherein, A is an interleukin 2 (IL-2) polypeptide; B is a half-lifeextension element, wherein the half-life extension element is humanserum albumin or an antigen-binding polypeptide that binds human serumalbumin; L1 is a protease-cleavable polypeptide linker that comprises atleast one sequence that is cleavable by a protease, L2 is a polypeptidelinker that is optionally protease-cleavable, and whenprotease-cleavable L2 comprises at least one sequence that is cleavableby a protease, wherein for each of L1 and L2, independently, theprotease is selected from the group consisting of a kallikrein,thrombin, chymase, carboxypeptidase A, an elastase, PR-3, granzyme M, acalpain, a matrix metalloproteinase (MMP), a fibroblast activationprotein (FAP), an ADAM metalloproteinase, a plasminogen activator, acathepsin, a caspase, a tryptase, and a tumor cell surface protease; andD is an IL-2 blocking moiety, wherein the blocking moiety is an antibodyor antigen-binding fragment of an antibody that binds the IL-2polypeptide; wherein the fusion polypeptide has attenuated IL-2-receptoractivating activity, wherein the IL-2-receptor activating activity ofthe fusion polypeptide is at least about 10 fold less than theIL-2-receptor activating activity of the polypeptide that comprises theIL-2 polypeptide that is produced by cleavage of the protease-cleavablepolypeptide linker L1 or, when L2 is protease-cleavable, by cleavage ofboth L1 and L2, and wherein the IL-2-receptor activating activity isassessed using a CTLL-2 proliferation assay, a phospho STAT ELISA, orHEK Blue reporter cell assay with equal amounts on a mole basis of theIL-2 polypeptide and the fusion polypeptide.
 2. The fusion polypeptideof claim 1, wherein the antibody fragment that binds the IL-2polypeptide is a single domain antibody, Fab or scFv.
 3. The fusionpolypeptide of claim 1, wherein L2 is a protease-cleavable polypeptidelinker.
 4. The fusion polypeptide of claim 1, wherein L1 or L2 or bothL1 and L2 are each cleaved by two or more different proteases.
 5. Thefusion polypeptide of claim 1, wherein the IL-2 blocking moiety inhibitsactivation of the IL-2 receptor by the fusion polypeptide.
 6. The fusionpolypeptide of claim 1, wherein the cathepsin is cathepsin B, cathepsinC, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G.7. The fusion polypeptide of claim 1, wherein the matrix metalloprotease(MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, orMMP14.
 8. A nucleic acid encoding the polypeptide of claim
 1. 9. Anisolated vector comprising the nucleic acid of claim
 8. 10. An isolatedhost cell comprising the vector of claim
 9. 11. A method of making apharmaceutical composition, comprising culturing the isolated host cellof claim 10 under suitable conditions for expression and collection ofthe fusion polypeptide.